Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P30/00—Technologies relating to oil refining and petrochemical industry
  • Y02P30/20—Technologies relating to oil refining and petrochemical industry using bio-feedstock

Definitions

• Crude petroleum is a very complex mixture containing a wide range of hydrocarbons. It is converted into a diversity of fuels and chemicals through a variety of chemical processes in refineries. Crude petroleum is a source of transportation fuels as well as a source of raw materials for producing petrochemicals. Petrochemicals are used to make specialty chemicals such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, and gels.
• Branched hydrocarbons, branched fatty acids and other branched chain fatty acid derivatives are known to have additional preferred properties when compared to straight- chain molecules of same molecular weight (i.e., isomers), such as considerably lower melting points which can in turn confer lower pour points when made into industrial chemicals.
• branched hydrocarbons, branched fatty acids, and other branched fatty acid derivates to confer substantially lower volatility and vapor pressure, and improved stability against oxidation and rancidity, thus making them particularly suited as components or feedstock of cosmetic and pharmaceutical applications, or as components of plasticizers for synthetic resins, solvents for solutions for printing ink and specialty inks, and industrial lubricants.
• Such additional preferred properties can also be obtained in unsaturated fatty acid derivatives (including unsaturated hydrocarbons, unsaturated fatty acids, and other unsaturated fatty acid derivates), typically with high degrees of unsaturation, but unsaturation promotes oxidation and can lead to short shelf lives and corrosion problems. Therefore, lower melting points, pour points, volatility, and vapor pressure, as well as improved oxidative stability, are better obtained through branching.
• unsaturated fatty acid derivatives including unsaturated hydrocarbons, unsaturated fatty acids, and other unsaturated fatty acid derivates
• branched specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of thermal energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals.
• branched chemicals such as, for example, branched alkanes, branched alkenes, branched fatty acids, branched fatty esters, branched fatty alcohols and branched fatty aldehydes by isomerization of straight-chain hydrocarbons, using various catalytic processes. Expensive catalysts are typically employed in these processes, therefore increasing the costs of manufacturing. The catalysts that are used often become undesirable contaminants that must be removed from the finished products, thus adding further costs to the processes.
• gasoline comprises predominantly straight chain , branched chain, and aromatic hydrocarbons ranging from about 4 to 12 carbon atoms
• diesel predominantly comprises straight chain hydrocarbons ranging from about 9 to 23 carbon atoms.
• Diesel fuel quality is evaluated by parameters such as cetane number, kinematic viscosity, oxidative stability, and cloud point (Knothe G., Fuel Process Technol. 86:1059-1070 (2005)). These parameters, among others, are impacted by the hydrocarbon chain length as well as by the degree of branching or saturation of the hydrocarbon.
• Microbially-produced fatty acids and other fatty acid derivatives can be readily tailored by genetic manipulation.
• Metabolic engineering enables microbial strains to produce different mixtures of fatty acids and other fatty acid derivatives, which can be optimized in meeting or exceeding fuel standards, and can be tailored to produce other chemicals or precursor molecules that are typically petroleum derived.
• the present invention provides novel recombinant microbial cells which produce branched chain fatty acid derivatives.
• the invention also provides methods of making branched chain fatty acid derivatives comprising culturing recombinant microbial cell of the invention, and other features apparent upon further review.
• the invention provides a recombinant microbial cell comprising: (a) polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched-chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydrolipoamide dehydrogenase activity, and (b) polynucleotides encoding a polypeptide having beta-ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate, wherein at least one polynucleotide according to (a) or (b) encodes a polypeptide that is exogenous to the recombinant microbial cell or expression of said polynucleotide is modulated in the recombinant microbial cell; and comprising one or more polynucleotides each which
• expression of the at least one polynucleotide according to (a) or (b) is modulated by overexpression of the polynucleotide, such as by operatively linking the polynucleotide to an exogenous promoter.
• the recombinant microbial cell according to the first aspect produces a fatty acid derivative composition when the cell is cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides, the fatty acid derivative composition comprising straight chain fatty acid derivatives and branched chain fatty acid derivatives, and the branched chain fatty acid derivatives comprising anteiso-branched fatty acid derivatives and iso-branched fatty acid derivatives.
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty acid derivatives in the composition produced by the microbial cell of the first aspect are branched chain fatty acid derivatives. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty acid derivatives in the composition produced by the microbial cell of the first aspect are iso-branched fatty acid derivatives.
• At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched fatty acid derivatives in the composition produced by the microbial cell of the first aspect are anteiso-branched fatty acid derivatives.
• the recombinant microbial cell of the first aspect produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty acid derivatives when cultured in a culture medium containing a carbon source under conditions effective to express the
• the invention provides a recombinant microbial cell comprising: (a) polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched-chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydrolipoamide dehydrogenase activity, and (b) polynucleotides encoding a polypeptide having beta-ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate, and further comprising : (c) polynucleotides encoding polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deamina
• BKD branched chain alpha-ket
• expression of the at least one polynucleotide according to (a), (b), (c), (d), or (e) is modulated by overexpression of the polynucleotide, such as by operatively linking the polynucleotide to an exogenous promoter.
• the recombinant microbial cell according to the second aspect produces a fatty acid derivative composition when the cell is cultured in a culture medium containing a carbon source under conditions effective to express the
• the fatty acid derivative composition comprising straight chain fatty acid derivatives and branched chain fatty acid derivatives, the branched chain fatty acid derivatives comprising anteiso-branched fatty acid derivatives and iso-branched fatty acid derivatives.
• at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty acid derivatives in the composition produced by the recombinant microbial cell according to the second aspect are branched chain fatty acid derivatives.
• At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty acid derivatives in the composition produced by the microbial cell of the second aspect are anteiso-branched fatty acid derivatives.
• the recombinant microbial cell of the second aspect produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty acid derivatives when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative enzyme activity comprises thioesterase activity
• the branched chain fatty acid derivative produced by the recombinant microbial cell is a branched chain fatty acid.
• the recombinant microbial cell produces a fatty acid composition comprising straight chain fatty acids and branched chain fatty acids, the branched chain fatty acids comprising anteiso-branched fatty acids and iso-branched fatty acids .
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty acids in the composition are branched fatty acids. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty acids in the composition produced by the microbial cell are anteiso-branched fatty acids.
• the recombinant microbial cell produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty acids or anteiso- branched chain fatty acids when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative enzyme activity comprises ester synthase activity
• the branched chain fatty acid derivative produced by the recombinant microbial cell is a branched fatty ester.
• the recombinant microbial cell produces a fatty ester composition comprising straight chain fatty esters and branched chain fatty esters, the branched chain esters comprising anteiso-branched fatty esters and iso-branched fatty esters.
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty esters in the composition are branched fatty esters .
• at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched fatty esters in the composition produced by the microbial cell are anteiso- branched fatty esters or iso-branched fatty esters .
• the recombinant microbial cell produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty esters or anteiso-branched fatty esters or iso-branched fatty esters when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative enzyme activity comprises fatty aldehyde biosynthesis activity
• the branched chain fatty acid derivative produced by the recombinant microbial cell is a branched fatty aldehyde.
• the recombinant microbial cell produces a fatty aldehyde composition comprising straight chain fatty aldehydes and branched chain fatty aldehydes, the branched fatty aldehydes comprising anteiso-branched fatty aldehydes and iso- branched fatty aldehydes.
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty aldehydes in the composition are branched fatty aldehydes.
• At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty aldehydes in the composition produced by the microbial cell are anteiso-branched fatty aldehydes or iso- branched fatty aldehydes.
• the recombinant microbial cell produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty aldehydes or anteiso-branched fatty aldehydes or iso-branched fatty aldehydes when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative enzyme activity comprises fatty alcohol biosynthesis activity
• the branched chain fatty acid derivative produced by the recombinant microbial cell is a branched fatty alcohol.
• the recombinant microbial cell produces a fatty alcohol composition comprising straight chain fatty alcohols and branched chain fatty alcohols, the branched fatty alcohols comprising anteiso-branched fatty alcohols and iso-branched fatty alcohols.
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty alcohols in the composition are branched fatty alcohols .
• at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty alcohols in the composition produced by the microbial cell are anteiso-branched fatty alcohols or iso-branched fatty alcohols .
• the recombinant microbial cell produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty alcohols or anteiso-branched fatty alcohols or iso-branched fatty alcohols when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative enzyme activity comprises hydrocarbon biosynthesis activity
• the branched chain fatty acid derivative produced by the recombinant microbial cell is a branched hydrocarbon, such as a branched alkane, a branched terminal olefin or a branched internal olefin.
• the recombinant microbial cell produces a hydrocarbon composition comprising straight chain hydrocarbons and branched chain hydrocarbons , the branched hydrocarbons comprising anteiso-branched hydrocarbons and iso-branched hydrocarbons .
• At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the hydrocarbons in the composition are branched hydrocarbons .
• at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain hydrocarbons in the composition produced by the microbial cell are anteiso-branched hydrocarbons or iso-branched hydrocarbons.
• the recombinant microbial cell produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched hydrocarbons or anteiso-branched hydrocarbons or iso-branched hydrocarbons when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the carbon source comprises a monosaccharide, a disaccharide, or an oligosaccharide.
• the carbon source comprises a monosaccharide, preferably a hexose or a pentose , preferably a hexose such as glucose.
• the carbon source is obtained from biomass, such as a cellulosic hydrolysate.
• the carbon source comprises a branched short-chain carboxylic acid such as isobutyrate, isovalerate, or 2-methyl-butyrate.
• the host (e.g., parental) microbial cell is a filamentous fungi, an algae, a yeast, or a prokaryote such as a bacteria.
• the host cell is a bacterial cell.
• the host cell is an E. coli cell or a Bacillus cell, preferably an E. coli cell.
• the recombinant microbial cell according to the first aspect or the second aspect comprises a polynucleotide encoding a polypeptide having branched- chain alpha-keto acid dehydrogenase activity which is categorized as EC 1.2.4.4.
• the polypeptide having branched-chain alpha-keto acid dehydrogenase activity has an alpha subunit and a beta subunit, encoded by a bkdA and bkdB gene, a bkdAA and bkdAB gene, or a Pput_1453 and Pput_1452 gene.
• the polypeptide having branched-chain alpha-keto acid dehydrogenase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having branched- chain alpha-keto acid dehydrogenase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is
• the polypeptide having branched-chain alpha-keto acid dehydrogenase activity comprises an alpha subunit and a beta subunit.
• the alpha subunit comprises a sequence selected from SEQ ID NOs:l, 3, 5, 7, 9, 11, and 13, or a fragment or a variant thereof, or comprises one or more motif selected from SEQ ID NOs:15-21, wherein the alpha subunit combined with a beta subunit has branched-chain alpha-keto acid dehydrogenase activity, and which, when combined with a polypeptide having lipoamide acyltransferase activity and a polypeptide having dihydrolipoamide dehydrogenase activity, catalyzes the conversion of a branched alpha-keto acid to a branched acyl-CoA in vitro or in vivo, preferably in vivo.
• the beta subunit comprises a sequence selected from SEQ ID NOs:22, 24, 26, 28, 30, 32, and 34, or a fragment or a variant thereof, or comprises one or more motif selected from SEQ ID NOs:36-42, wherein the beta subunit combined with an alpha subunit has branched-chain alpha-keto acid dehydrogenase activity, and which, when combined with a polypeptide having lipoamide acyltransferase activity and a polypeptide having dihydrolipoamide dehydrogenase activity, catalyzes the conversion of a branched alpha-keto acid to a branched acyl-CoA in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the first aspect or the second aspect comprises a polynucleotide encoding a polypeptide having lipoamide acyltransferase activity which is categorized as EC 2.3.1.168.
• the polypeptide having lipoamide acyltransferase activity is encoded by a bkdB, a bkdC, or a Pput_1451 gene.
• the polypeptide having lipoamide acyltransferase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having lipoamide acyltransferase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the
• the polypeptide having lipoamide acyltransferase activity comprises a sequence selected from SEQ ID NOs:43, 45, 47, 49, 51, 53, and 55, or a variant or a fragment thereof having lipoamide acyltransferase activity, or comprises one or more sequence motif selected from SEQ ID NOs:57-62, and which, when combined with a polypeptide having dihydrolipoamide dehydrogenase activity and a polypeptide having branched-chain alpha-keto acid dehydrogenase activity, catalyzes the conversion of a branched alpha-keto acid to a branched acyl-CoA in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the first aspect or the second aspect comprises a polynucleotide encoding a polypeptide having lipoamide dehydrogenase activity which is categorized as EC 1.8.1.4.
• the polypeptide having dihydrolipoamide dehydrogenase activity is encoded by a IpdV gene, a Pput_1450 gene or a IpdA gene.
• dihydrolipoamide dehydrogenase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having dihydrolipoamide dehydrogenase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having dihydrolipoamide dehydrogenase activity comprises a sequence selected from SEQ ID NOs:63, 65, 67, 69, 71, 73, 75, and 77, or a variant or a fragment thereof having dihydrolipoamide dehydrogenase activity, or comprises one or more sequence motif selected from SEQ ID NOs:79-83 and which, when combined with a polypeptide having lipoamide acyltransferase activity and a polypeptide having branched-chain alpha-keto acid dehydrogenase activity, catalyzes the conversion of a branched alpha-keto acid to a branched acyl-CoA in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the first aspect or the second aspect comprises a polynucleotide encoding a polypeptide having beta- ketoacyl-ACP synthase activity and utilizes a branched acyl-CoA molecule as a substrate, preferably a beta-ketoacyl-ACP synthase III activity categorized as EC 2.3.1.180.
• the polypeptide having beta-ketoacyl-ACP synthase activity is encoded by a fabH gene.
• the polypeptide having beta-ketoacyl-ACP synthase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having beta-ketoacyl-ACP synthase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to a strong promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having beta-ketoacyl-ACP synthase activity comprises a sequence selected from SEQ ID NOs:84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, and 108, or a variant or a fragment thereof having beta-ketoacyl-ACP synthase activity, or comprises one or more sequence motif selected from SEQ ID NO s:110-115, and which catalyzes the condensation of a branched acyl-CoA with malonyl-ACP to form a branched acyl-ACP in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the first aspect or the second aspect comprises an endogenous polynucleotide sequence (such as, an endogenous fabH gene) encoding a polypeptide having beta-ketoacyl-ACP synthase activity that does not utilize a branched acyl-CoA molecule as a substrate, and expression of the endogenous polynucleotide sequence in the recombinant microbial cell is attenuated. In some embodiments, expression of the endogenous polynucleotide is attenuated by deletion of all or part of the sequence of the endogenous polynucleotide in the recombinant microbial cell.
• an endogenous polynucleotide sequence such as, an endogenous fabH gene
• expression of the endogenous polynucleotide sequence in the recombinant microbial cell is attenuated.
• expression of the endogenous polynucleotide is atten
• the recombinant microbial cell according to the first aspect or the second aspect comprises an endogenous polynucleotide sequence (such as, an endogenous fadE gene) encoding a polypeptide having acyl-CoA dehydrogenase activity, and expression of the endogenous polynucleotide in the recombinant microbial cell is attenuated.
• expression of the endogenous polynucleotide is attenuated by deletion of all or part of the sequence of the endogenous polynucleotide in the recombinant microbial cell.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having aspartokinase activity which is categorized as EC 2.7.2.4.
• the polypeptide having aspartokinase activity is encoded by a thrA, a dapG or a hom3 gene.
• the polypeptide having aspartokinase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having aspartokinase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the
• polypeptide having aspartokinase activity comprises a sequence selected from SEQ. ID NOs:116,118,120,122,124, or a variant or a fragment thereof having aspartokinase activity and which catalyzes the conversion of aspartate to aspartyl phosphate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having homoserine dehydrogenase activity which is categorized as EC 1.1.1.3.
• the polypeptide having homoserine dehydrogenase activity is encoded by a thrA, a horn or a hom6 gene.
• the polypeptide having homoserine dehydrogenase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• homoserine dehydrogenase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• homoserine dehydrogenase activity comprises a sequence selected from SEQ ID NOs:116, 118, 126, 128, and 130, or a variant or a fragment thereof having homoserine
• dehydrogenase activity and which catalyzes the conversion of aspartate semialdehyde to homoserine in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having aspartokinase and homoserine dehydrogenase activity.
• the polypeptide having aspartokinase and homoserine dehydrogenase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having aspartokinase and homoserine dehydrogenase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having aspartokinase and homoserine dehydrogenase activity comprises the sequence SEQ ID NO:116, or a variant or a fragment thereof, such as SEQ ID NO:118, which catalyzes the conversion of aspartate to aspartyl phosphate and the conversion of aspartate semialdehyde to homoserine in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having homoserine kinase activity which is categorized as EC 2.7.1.39.
• the polypeptide having homoserine kinase activity is encoded by a thrB gene or a thrl gene.
• the polypeptide having homoserine kinase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having homoserine kinase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having homoserine kinase activity comprises a sequence selected from SEQ ID NOs:132,134,136,138, or a variant or a fragment thereof having homoserine kinase activity and which catalyzes the conversion of homoserine to O- phospho-L-homoserine in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having threonine synthase activity which is categorized as EC 4.2.3.1.
• the polypeptide having threonine synthase activity is encoded by a thrC gene.
• the polypeptide having threonine synthase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having threonine synthase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having threonine synthase activity comprises a sequence selected from SEQ ID Os: 140, 143, 144, or a variant or a fragment thereof having threonine synthase activity and which catalyzes the conversion of O-phospho-L-homoserine to threonine in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having threonine deaminase activity which is categorized as EC 4.3.1.19.
• the polypeptide having threonine deaminase activity is encoded by a tdcB gene or an ilvA gene.
• the polypeptide having threonine deaminase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having threonine deaminase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having threonine deaminase activity comprises a sequence selected from SEQ.
• ID NOs:146,148,150,152, and 154 or a variant or a fragment thereof having threonine deaminase activity and which catalyzes the conversion of threonine to 2-ketobutyrate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having (R)-citramalate synthase activity which is categorized as EC 2.3.1.182.
• the polypeptide having (R)- citramalate synthase activity is encoded by a cimA gene.
• the polypeptide having (R)-citramalate synthase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having (R)-citramalate synthase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having (R)-citramalate synthase activity comprises a sequence selected from SEQ ID NOs:156,158,160, and 162, or a variant or a fragment thereof having (R)-citramalate synthase activity and which catalyzes the reaction of acetyl- CoA and pyruvate to (R)-citramalate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having isopropylmalate isomerase activity which is categorized as EC 4.2.1.33.
• the polypeptide having isopropylmalate isomerase activity comprises a large subunit and a small subunit encoded by leuCD genes.
• the polypeptide having isopropylmalate isomerase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having isopropylmalate isomerase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the
• polypeptide having isopropylmalate isomerase activity comprises a large subunit and a small subunit.
• the polypeptide having isopropylmalate isomerase activity comprises a large subunit sequence selected from SEQ. ID NOs:164 and 168 and a small subunit sequence selected from SEQ ID NOs:166 and 170, or variants or fragments thereof having isopropylmalate isomerase activity and which catalyzes the conversion of (R)- citramalate to citraconate and citraconate to beta-methyl-D-malate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having beta-isopropylmalate
• the polypeptide having beta-isopropyl malate dehydrogenase activity which is categorized as EC 1.1.1.85.
• the polypeptide having beta-isopropyl malate dehydrogenase activity is encoded by a leuB gene or a Ieu2 gene.
• the polypeptide having beta-isopropylmalate dehydrogenase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having beta-isopropylmalate dehydrogenase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having beta-isopropyl malate dehydrogenase activity comprises a sequence selected from SEQ ID NOs:172,174,176, or a variant or a fragment thereof having beta-isopropylmalate dehydrogenase activity and which catalyzes conversion of beta-methyl-D-malate to 2-ketobutyrate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having acetohydroxyacid synthase activity which is categorized as EC 2.2.1.6.
• the polypeptide having acetohydroxyacid synthase activity comprises a large subunit and a small subunit encoded by ilvBN genes, ilvGM genes or ilvlH genes.
• the polypeptide having acetohydroxyacid synthase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having acetohydroxyacid synthase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having acetohydroxyacid synthase activity comprises a sequence selected from SEQ ID NOs:178, 180, 182, 184, 186, 188, 190, and 192, or a variant or a fragment thereof having acetohydroxyacid synthase activity and which catalyzes the conversion of 2-ketobutyrate to 2-keto-3-methylvalerate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having acetohydroxyacid
• polypeptide having acetohydroxyacid isomeroreductase activity which is categorized as EC 1.1.1.86.
• the polypeptide having acetohydroxyacid isomeroreductase activity is encoded by an //vC gene or an ilv5 gene.
• isomeroreductase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having acetohydroxyacid isomeroreductase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide. is overexpressed in the recombinant microbial cell.
• the polypeptide having acetohydroxyacid isomeroreductase activity comprises a sequence selected from SEQ.
• ID NOs:194 and 196 or a variant or a fragment thereof having acetohydroxyacid isomeroreductase activity and which catalyzes the conversion of 2- aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-methylvalerate in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the second aspect comprises a polynucleotide encoding a polypeptide having dihydroxy acid dehydratase activity which is categorized as EC 4.2.1.9.
• the polypeptide having acetohydroxyacid isomeroreductase activity is encoded by an ilvD gene or an ilv3 gene.
• the polypeptide having dihydroxy acid dehydratase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having dihydroxy acid dehydratase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having dihydroxy acid dehydratase activity comprises a sequence selected from SEQ ID NO:198 and 200, or a variant or a fragment thereof having dihydroxy acid dehydratase activity and which catalyzes the conversion of 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate in vitro or in vivo, preferably in vivo.
• a recombinant microbial cell according to the first aspect or the second aspect further comprises one or more polynucleotides encoding one or more polypeptides each having a fatty acid derivative enzyme activity, wherein the recombinant microbial cell produces a branched chain fatty acid derivative when cultured in the presence of a carbon source.
• the fatty acid derivative enzyme activity comprises a thioesterase activity, an ester synthase activity, a fatty aldehyde biosynthesis activity, a fatty alcohol biosynthesis activity, a ketone biosynthesis activity, and/or a hydrocarbon biosynthesis activity.
• the recombinant microbial cell comprises polynucleotides encoding two or more polypeptides, each polypeptide having a fatty acid derivative enzyme activity.
• the recombinant microbial cell expresses or overexpresses one or more polypeptides having fatty acid derivative enzyme activity selected from: (1) a polypeptide having thioesterase activity; (2) a polypeptide having decarboxylase activity; (3)a polypeptide having carboxylic acid reductase activity; (4) a polypeptide having alcohol dehydrogenase activity (EC 1.1.1.1); (5) a polypeptide having aldehyde decarbonylase activity (EC 4.1.99.5); (6) a polypeptide having acyl-CoA reductase activity (EC 1.2.1.50); (7) a polypeptide having acyl-ACP reductase activity; (8) a polypeptide having ester synthase activity (EC 3.1.1.67); (9) a polypeptide having OleA activity; or (10) a polypeptide having OleCD or OleBCD activity; wherein the recombinant microbial cell produces a composition comprising
• the fatty acid derivative enzyme activity is a thioesterase activity
• the branched chain fatty acid derivative is a branched chain fatty acid, wherein a culture comprising the recombinant microbial cell produces a branched chain fatty acid composition when cultured in the presence of a carbon source.
• the polypeptide has a thioesterase activity which is categorized as EC 3.1.1.5, EC 3.1.2.- , or EC 3.1.2.14.
• the polypeptide having a thioesterase activity is encoded by a tesA , a tesB , a fotA, or a fatB gene.
• the polypeptide having thioesterase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having thioesterase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter, such that the polynucleotide is overexpressed in the recombinant microbial cell.
• the polypeptide having thioesterase activity is endogenous to the parental microbial cell, or is exogenous to the parental microbial cell.
• expression of the polynucleotide encoding the polypeptide having thioesterase activity is modulated in the recombinant microbial cell.
• expression of the polynucleotide is modulated by operatively linking the polynucleotide to an exogenous promoter,
• thioesterase activity comprises a sequence selected from SEQ ID NO: 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, and 224, or a variant or a fragment thereof having thioesterase activity and which catalyzes the hydrolysis of a branched acyl-ACP to a branched fatty acid, or catalyzes the alcoholysis of a branched acyl-ACP to a branched fatty ester, in vitro or in vivo, preferably in vivo.
• the recombinant microbial cell according to the first aspect or the second aspect further comprising a polynucleotide encoding a polypeptide having thioesterase activity, when cultured in the presence of a carbon source, produces at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L branched chain fatty acids or anteiso-branched chain fatty acids when cultured in a culture medium containing a carbon source under conditions effective to express the polynucleotides.
• the recombinant microbial cell according to the first aspect or the second aspect, further comprising a polynucleotide encoding a polypeptide having thioesterase activity produces a fatty acid composition comprising straight chain fatty acids and branched chain fatty acids, the branched chain fatty acids comprising anteiso-branched fatty acids and iso-branched fatty acids .
• at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the fatty acids in the composition are branched fatty acids.
• At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the branched chain fatty acids in the composition produced by the microbial cell are anteiso-branched fatty acids.
• the invention includes a method of making a composition comprising a branched fatty acid derivative, the method comprising: obtaining a
• recombinant microbial cell comprising : (a) polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched- chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydrolipoamide dehydrogenase activity, and (b) a polynucleotide encoding a polypeptide having beta-ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate, wherein at least one polynucleotide according to (a) or (b) encodes a polypeptide that is exogenous to the parental microbial cell or expression of said polynucleotide is modulated in the recombinant microbial cell; the recombinant microbial cell further comprising one or more polynucleotides each which encode
• the fatty acid derivative composition produced by the recombinant cell comprises branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight of the fatty acid derivatives in the composition are branched fatty acid derivatives.
• the fatty acid derivative composition comprises branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L .
• the fatty acid derivative enzyme activity comprises a thioesterase activity, an ester synthase activity, a fatty aldehyde biosynthesis activity, a fatty alcohol biosynthesis activity, a ketone biosynthesis activity, and/or a hydrocarbon biosynthesis activity.
• the recombinant microbial cell comprises polynucleotides encoding two or more polypeptides, each polypeptide having a fatty acid derivative enzyme activity.
• the recombinant microbial cell expresses or overexpresses one or more polypeptides having fatty acid derivative enzyme activity selected from: (1) a polypeptide having thioesterase activity; (2) a polypeptide having decarboxylase activity; (3) a polypeptide having carboxylic acid reductase activity; (4) a polypeptide having alcohol dehydrogenase activity (EC 1.1.1.1); (5) a polypeptide having aldehyde decarbonylase activity (EC 4.1.99.5); (6) a polypeptide having acyl-CoA reductase activity (EC 1.2.1.50); (7) a polypeptide having acyl-ACP reductase activity; (8) a polypeptide having ester synthase activity (EC 3.1.1.67); (9) a polypeptide having OleA activity; or (10) a polypeptide having OleCD or OleBCD activity; wherein the recombinant microbial cell produces a composition comprising
• the fatty acid derivative composition produced by the recombinant cell comprises branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight of the fatty acid derivatives in the composition are branched fatty acid derivatives.
• the fatty acid derivative composition comprises branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L .
• the fatty acid derivative composition produced by the recombinant microbial cell culture comprises iso-branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% by weight of the branched fatty acid derivatives in the composition are iso- branched fatty acid derivatives.
• the invention includes a method of making a composition comprising an anteiso-branched fatty acid derivative, the method comprising: obtaining a recombinant microbial cell (such as, a culture comprising a recombinant microbial cell) comprising : (a) polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched-chain alpha-keto acid
• a recombinant microbial cell such as, a culture comprising a recombinant microbial cell
• BKD branched chain alpha-keto acid dehydrogenase
• a polynucleotide encoding a polypeptide having beta- ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate comprising (c) polynucleotides encoding polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deaminase activity, or (d) polynucleotides encoding polypeptides having (R)- citramalate synthase activity , isopropylmalate isomerase activity, and beta-isopropyl malate dehydrogenase activity, or (c) and (d); and (e) polypeptides having acetohydroxyacid synthase activity, acetohydroxyacid isomeroreductase activity, and dihydroxy acid
• the fatty acid derivative enzyme activity comprises a thioesterase activity, an ester synthase activity, a fatty aldehyde biosynthesis activity, a fatty alcohol biosynthesis activity, a ketone biosynthesis activity and/or a hydrocarbon biosynthesis activity, as described hereinabove; wherein the recombinant microbial cell produces a composition comprising anteiso-branched fatty acids, anteiso-branched fatty esters, anteiso-branched wax esters, anteiso-branched fatty aldehydes, anteiso-branched fatty alcohols, anteiso-branched alkanes, anteiso-branched alkenes, anteiso-branched terminal olefins, or anteiso-branched ketones.
• the fatty acid derivative composition produced by the recombinant cell comprises branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight of the fatty acid derivatives in the composition are branched fatty acid derivatives.
• the fatty acid derivative composition comprises branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L .
• the fatty acid derivative composition produced by the recombinant microbial cell culture comprises anteiso-branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% by weight of the branched fatty acid derivatives in the composition are anteiso-branched fatty acid derivatives.
• the fatty acid derivative composition comprises anteiso-branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 25 mg/L, at least 100 mg/L, at least 200 mg/L, at least 500 mg/L, at least 1000 mg/L, or at least 2000 mg/L .
• an amount e.g., a titer
• Figure 1 depicts a branched chain fatty acid (BCFA) biosynthetic pathway as described herein.
• BCFA branched chain fatty acid
• Figures 2A and 2B depict exemplary intermediates and products of the BCFA biosynthetic pathway when supplied with the branched acyl-CoA molecules
• A 2-methyl-butyryi-CoA , which generates the anteiso-branched ⁇ -ketoacyl-ACP intermediate 4-methyl-3-oxo- hexanoyl-ACP, leading to production of anteiso-branched fatty acid derivatives
• B isobutyryl-CoA, which generates the iso-branched ⁇ -ketoacyl-ACP intermediate 4-methyl-3- oxo-pentanoyl-ACP, leading to production of iso-branched fatty acid derivatives.
• the branched acyl-CoA molecule isovaleryl-CoA can produce iso-branched fatty acid derivatives via the iso-branched ⁇ -ketoacyl-ACP intermediate 5-methyl-3-oxo-hexanoyl-ACP.
• Figures 3A and 3B depicts an anteiso-BCFA biosynthetic pathway as described herein.
• Figure 4 shows representative GC-MS traces of (a) fatty acids produced by an E. coli strain expressing a leaderless TesA polypeptide and B. subtilis FabHl protein, compared to (b) fatty acids produced by the same E. coli strain which does not express the B. subtilis FabH l protein.
• Figure 6 shows a representative GC/MS trace of fatty acids produced by an E. coli strain expressing a leaderless TesA polypeptide, P. putida BKD complex and B. subtilis FabHl.
• the peaks corresponding to iso-branched ("i-"), anteiso-branched (“a-"), monounsaturated (Cn:l) and saturated (Cn:0) fatty acids of chain length (Cn) from C13 to C17 are labeled accordingly.
• Figure 7 shows representative GC/MS traces of fatty alcohols produced by f. coli strains expressing (A) S. elongatus AAR , plus plasmids expressing the P. putida BKD complex and B. subtilis FabHl, and (B) the same E. coli strain expressing 5. elongatus AAR but lacking the plasmids expressing BKD and FabHl. Peaks representing branched fatty alcohols are boxed.
• NCBI Accession Numbers are identified herein as "NCBI Accession Numbers", or alternatively as “GenBank Accession Numbers”
• UniProtKB Accession Numbers are identified herein as "UniProtKB Accession Numbers”
• sequence identified by an NCBI / Gen Bank Accession number is version number 1 (that is, the Version Number of the sequence is "AccessionNumber.l” ).
• the NCBI and UniProtKB accession numbers provided herein were those current as of March 31, 2011.
• EC numbers classify enzymes according to the reaction catalyzed.
• EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, EC numbers are as provided in the KEGG database as of March 31, 2011.
• R represents an aliphatic group, preferably an alkyl group, which can comprise between about 4 and about 22 carbon atoms.
• Fatty acids can be saturated,
• R can have one or more points of unsaturation.
• R can be a straight chain or a branched chain.
• the branched chain may have one or more points of branching.
• the branched chain can have an iso- or anteiso- conformation.
• branched fatty acid is synonymous with “branched chain fatty acid” and is abbreviated “BCFA” herein.
• branched fatty acid derivative is synonymous with “branched chain fatty acid derivative” and is abbreviated herein "BCFA derivative”.
• branched fatty aldehyde is synonymous with “branched fatty acid aldehyde”, “branched chain fatty aldehyde” and “branched chain fatty acid aldehyde”;
• branched fatty alcohol is synonymous with “branched fatty acid alcohol”, “branched chain fatty alcohol” and “branched chain fatty acid alcohol”;
• branched fatty ester is synonymous with “branched fatty acid ester”, “branched chain fatty ester” and “branched chain fatty acid ester”; and so on.
• branched hydrocarbon is synonymous with “branched chain hydrocarbon”
• branched alkane is synonymous with “branched chain alkane”, and so on.
• an "iso-" branched chain refers to a branched hydrocarbon structure having a methyl on the penultimate carbon atom
• an "anteiso-" branched chain refers to a branched hydrocarbon structure having a methyl on the third carbon atom from the end.
• branched ⁇ -ketoacyl-ACP refers to the product of the condensation of a branched acyl-CoA "primer” molecule with malonyl-ACP catalyzed by a beta ketoacyl-ACP synthase III (i.e., FabH) enzyme as represented by part (D) of the BCFA pathway in Figures 1, 2 and 3.
• FabH beta ketoacyl-ACP synthase III
• This initial branched ⁇ -ketoacyl-ACP molecule enters the fatty acid synthase (FAS) cycle, represented by part (E) of Figure 1, where it is subjected to a round of keto reduction, dehydration, and enoyl reduction, forming a branched acyl-ACP molecule which then condenses with another malonyl-ACP molecule followed by another cycle of keto reduction, dehydration, and enoyl reduction, elongating the acyl chain of the branched acyl-ACP by two carbon units per cycle.
• FES fatty acid synthase
• the "branched acyl-ACP" elongation product is an acyl thioester formed between the carbonyl carbon of a branched alkyl chain and the sulfydryl group of the 4'-phosphopantethionyl moiety of an acyl carrier protein (ACP) and, as used herein, typically has the formula R-C(0)S-ACP, wherein R is a branched alkyl group which may be in the "iso-" or the "anteiso-" configuration.
• the branched acyl- ACP is an intermediate in the production of branched chain fatty acids and branched chain fatty acid derivatives by the BCFA pathways described herein.
• a "fatty acid derivative” is intended to include any product made at least in part by the fatty acid biosynthetic pathway of the recombinant microbial cell.
• a fatty acid derivative also includes any product made at least in part by a fatty acid pathway intermediate, such as an acyl-ACP intermediate.
• the fatty acid biosynthetic pathways described herein can include fatty acid pathway enzymes which can be engineered to produce fatty acid derivatives, and in some instances additional enzymes can be expressed to produce fatty acid derivatives having desired carbon chain characteristics, such as, for example, branched chain fatty acids and branched fatty acid derivatives (including, for example, anteiso-branched fatty acids and derivatives thereof) produced by enzymes of the branched chain fatty acid biosynthetic pathways described herein.
• Fatty acid derivatives include, but are not limited to, fatty acids, fatty aldehydes, fatty alcohols, fatty esters (such as waxes), hydrocarbons (such as alkanes and alkenes (e.g., terminal olefins and internal olefins)), and ketones.
• branched fatty acid derivative is intended to include any product made at least in part by a branched-chain fatty acid biosynthetic pathway (i.e., BCFA pathway) of a recombinant microbial cell described herein.
• a branched fatty acid derivative also includes any product made at least in part from a BCFA pathway intermediate, such as a branched acyl-ACP intermediate.
• Branched fatty acid derivatives include, but are not limited to, branched fatty acids, branched fatty aldehydes, branched fatty alcohols, branched fatty esters (such as waxes), branched hydrocarbons (such as branched alkanes and branched alkenes (e.g., branched terminal olefins and branched internal olefins)) and branched ketones.
• an “endogenous” polypeptide refers to a polypeptide encoded by the genome of the parental (i.e., host) cell from which the recombinant cell is engineered.
• exogenous polypeptide refers to a polypeptide which is not encoded by the genome of the parental microbial cell.
• variant (i.e., mutant) polypeptide is an example of an exogenous polypeptide.
• the endogenous polypeptide is overexpressed.
• Overexpression can be achieved by any suitable means.
• overexpress means to express or cause to be expressed a polynucleotide or a polypeptide in a cell at a greater concentration than is normally expressed in a corresponding host (for example, wild-type) cell under the same conditions.
• a polynucleotide can be "overexpressed" in a recombinant microbial cell when the polynucleotide is present in a greater concentration in the recombinant microbial cell as compared to its concentration in a non-recombinant microbial cell of the same species under the same conditions.
• increasing the level of expression of an endogenous polypeptide means to cause the overexpression of a polynucleotide sequence encoding the endogenous polypeptide, or to cause the overexpression of an endogenous polypeptide sequence.
• the degree of overexpression can be about 1.5-fold or more, about 2-fold or more, about 3-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 50-fold or more, about 100-fold or more, or any range therein.
• increasing the level of activity of an endogenous polypeptide means to enhance the biochemical or biological function (e.g., enzymatic activity) of an endogenous polypeptide.
• the degree of enhanced activity can be about 10% or more, about 20% or more, about 50% or more, about 75% or more, about 100% or more, about 200% or more, about 500% or more, about 1000% or more, or any range therein.
• exogenous regulatory element generally refers to a regulatory element (such as, an expression control sequence or a chemical compound) originating outside of the host cell.
• exogenous regulatory element e.g., "exogenous promoter”
• exogenous regulatory element can refer to a regulatory element derived from the host cell whose function is replicated or usurped for the purpose of controlling the expression of the endogenous polypeptide in the recombinant cell. For example, if the host cell is an E.
• the polypeptide is an endogenous polypeptide
• expression of the endogenous polypeptide the recombinant cell can be controlled by a promoter derived from another E. coli gene.
• the exogenous regulatory element that causes an increase in the level of expression and/or activity of an endogenous polypeptide is a chemical compound, such as a small molecule.
• the exogenous regulatory element which controls the expression of a polynucleotide e.g., an endogenous polynucleotide
• an expression control sequence which is operably linked to the endogenous polynucleotide by recombinant integration into the genome of the host cell.
• the expression control sequence is integrated into a host cell chromosome by homologous recombination using methods known in the art (e.g., Datsenko et al., Proc. Natl. Acad. Sci. U.S.A., 97(12): 6640-6645 (2000)).
• Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)).
• an expression control sequence is operably linked to a polynucleotide sequence.
• operably linked is meant that a polynucleotide sequence and expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s).
• Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation.
• Operably linked enhancers can be located upstream, within, or
• nucleic acid sequences such as nucleic acid sequences encoding selection markers, purification moieties, targeting proteins, and the like, can be operatively linked to the polynucleotide sequence, such that the additional nucleic acid sequences are expressed together with the polynucleotide sequence.
• the polynucleotide sequence is provided to the recombinant cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence.
• the promoter is a developmentally- regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell- specific promoter.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked.
• a useful vector is an episome (i.e., a nucleic acid capable of extra- chromosomal replication).
• Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as
• expression vectors In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome.
• plasmid and vector are used interchangeably herein, inasmuch as a plasmid is the most commonly used form of vector.
• vector also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.
• the recombinant vector comprises at least one sequence selected from the group consisting of (a) an expression control sequence operatively linked to the polynucleotide sequence; (b) a selection marker operatively linked to the
• polynucleotide sequence (c) a marker sequence operatively linked to the polynucleotide sequence; (d) a purification moiety operatively linked to the polynucleotide sequence; (e) a secretion sequence operatively linked to the polynucleotide sequence; and (f) a targeting sequence operatively linked to the polynucleotide sequence.
• the expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
• the expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.
• Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy- terminus of the recombinant polypeptide.
• Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
• a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide.
• enzymes include Factor Xa, thrombin, and enterokinase.
• Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, NJ; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA), and pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.
• GST glutathione S- transferase
• Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
• transformation and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).
• a gene that encodes a selectable marker e.g., resistance to an antibiotic
• selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin,
• Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Host cells which are stably transformed with the introduced nucleic acid, resulting in recombinant cells, can be identified by growth in the presence of an appropriate selection drug. Similarly, for stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.
• a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the gene of interest.
• selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and
• Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Host cells stably transfected with the introduced nucleic acid, resulting in recombinant cells, can be identified by growth in the presence of an appropriate selection drug.
• Gene knockout refers to a procedure by which a gene encoding a target protein is modified or inactivated so to reduce or eliminate the function of the intact protein. Inactivation of the gene may be performed by general methods such as
• mutagenesis by UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine, site-directed mutagenesis, homologous recombination, insertion-deletion mutagenesis, or "Red-driven integration" (Datsenko et ai, Proc. Natl. Acad. Sci. USA, 97:6640-45, 2000).
• a construct is introduced into a parental cell, such that it is possible to select for homologous recombination events in the resulting recombinant cell.
• knock-out construct including both positive and negative selection genes for efficiently selecting transfected (i.e., recombinant) cells that undergo a homologous recombination event with the construct.
• the alteration in the parental cell may be obtained, for example, by replacing through a single or double crossover recombination a wild type (i.e., endogenous) DNA sequence by a DNA sequence containing the alteration.
• the alteration may, for example, be a DNA sequence encoding an antibiotic resistance marker or a gene complementing a possible auxotrophy of the host cell. Mutations include, but are not limited to, deletion-insertion mutations.
• An example of such an alteration in a recombinant cell includes a gene disruption, i.e., a perturbation of a gene such that the product that is normally produced from this gene is not produced in a functional form. This could be due to a complete deletion, a deletion and insertion of a selective marker, an insertion of a selective marker, a frameshift mutation, an in-frame deletion, or a point mutation that leads to premature termination. In some instances, the entire mRNA for the gene is absent. In other situations, the amount of mRNA produced varies.
• an exogenous regulatory element which controls the expression of an endogenous polynucleotide is an expression control sequence which is operably linked to the endogenous polynucleotide by recombinant integration into the genome of the host cell.
• the expression control sequence is integrated into a host cell chromosome by homologous recombination using methods known in the art.
• the term "under conditions effective to express said polynucleotide sequences" means any conditions that allow a recombinant cell to produce a desired fatty acid derivative. Suitable conditions include, for example, fermentation conditions.
• Fermentation conditions can comprise many parameters, such as temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow.
• Exemplary culture media include broths or gels.
• the medium includes a carbon source that can be metabolized by a recombinant cell directly.
• Fermentation denotes the use of a carbon source by a production host, such as a recombinant microbial cell of the invention. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro-aerobic).
• a recombinant microbial cell can process a carbon source into a branched acyl-ACP or a desired branched fatty acid derivative (e.g., a branched fatty acid, branched fatty ester, branched fatty aldehyde, branched fatty alcohol, branched alkane, or branched olefin) will vary in part, based upon the specific microorganism.
• the process occurs in an aerobic environment.
• the process occurs in an anaerobic environment.
• the process occurs in a micro-aerobic environment.
• carbon source refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth.
• Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and C0 2 ).
• Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof.
• monosaccharides such as glucose, fructose, mannose, galactose, xylose, and arabinose
• oligosaccharides such as fructo-oligosaccharide
• the carbon source can be a branched short-chain carboxylic acid such as isobutyrate, isovalerate, or 2-methyl-butyrate.
• the carbon source can be a product of photosynthesis, such as glucose.
• the carbon source is biomass.
• the carbon source comprises sucrose.
• the carbon source comprises glucose.
• biomass refers to any biological material from which a carbon source is derived.
• a biomass is processed into a carbon source, which is suitable for bioconversion.
• the biomass does not require further processing into a carbon source.
• the carbon source can be converted into a biofuel.
• An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass.
• Another exemplary source of biomass is metabolic waste products, such as animal matter (e.g., cow manure).
• Further exemplary sources of biomass include algae and other marine plants.
• Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers.
• biomass also can refer to sources of carbon, such as carbohydrates (e.g.,
• a recombinant microbial cell can be cultured, for example, for about 4, 8, 12, 24, 36, 48, 72, or more hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow production or expression. For example, the recombinant microbial cells in the sample or the medium in which the recombinant microbial cells were grown can be tested for the presence of a desired product.
• a branched fatty acid When testing for the presence of a branched fatty acid, a branched fatty ester, a branched fatty aldehyde, a branched fatty alcohol, a branched hydrocarbon or other branched fatty acid derivative, assays, such as, but not limited to, gas chromatography (GC), mass spectroscopy (MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography (LC), GC coupled with a flame ionization detector (FID), GC-MS, and LC-MS can be used.
• assays such as, but not limited to, gas chromatography (GC), mass spectroscopy (MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography (LC), GC coupled with a flame ionization detector (FID), GC-MS, and LC-MS can be used.
• techniques such as, but not limited to, Western
• microorganism means prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, and higher Protista.
• microbes and microbial cells (i.e., cells from microbes) and are used interchangeably with
• microorganisms and refer to cells or small organisms that can only be seen with the aid of a microscope.
• the host cell is a microbial cell. In some embodiments, the host cell is selected from the genus Escherichia, Bacillus, Lactobacillus, Pantoea,
• Phanerochaete Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, Streptomyces, Synechococcus, Chlorella, or Prototheca.
• the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
• the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzoe cell, a Humicolo insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor mic ei cell.
• the host cell is a Streptomyces lividans cell or a
• the host cell is an Actinomycetes cell.
• the host cell is a Soccharomyces cerevisiae cell.
• the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cvl cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell.
• the host cell is a cell from an eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, an engineered organism thereof, or a synthetic organism.
• the host cell is light- dependent or fixes carbon.
• the host cell has autotrophic activity.
• the host cell has photoautotrophic activity, such as in the presence of light.
• the host cell is heterotrophic or mixotrophic in the absence of light.
• the host cell is a cell from Avabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlomydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp.
• PCC 6803 Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas jluorescens, Pantoea citrea or Zymomonas mobilis.
• the host cell is a cell from Chlorella fusca, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella kessleri, Chlorella vulgaris, Chlorella saccharophila, Chlorella sorokiniana, Chlorella ellipsoidea, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, or Prototheca zopfii.
• the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell.
• the host cell is an E. coli cell. In some embodiments, the host cell is an E. coli cell.
• the £ coli cell is a strain B, a strain C, a strain K, or a strain W £ coli cell.
• the host cell is engineered to express (or overexpress) a transport protein.
• Transport proteins can export polypeptides and organic compounds (e.g., fatty acids or derivatives thereof) out of a host cell.
• the term "metabolically engineered” or “metabolic engineering” involves rational pathway design and assembly of polynucleotides corresponding to biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired metabolite, such as, for example, a branched a-keto acid, a branched ⁇ -ketoacyl-ACP, a branched acyl-ACP, or a branched fatty acid derivative, in a recombinant cell, such as, a recombinant microbial cell.
• “Metabolically engineered” can further include optimization of metabolic flux by regulation and
• a "biosynthetic gene” can be native to the host cell (i.e., a gene which is not modified from the host cell), or, can be exogenous
• a biosynthetic gene encodes a "biosynthetic polypeptide" or a "biosynthetic enzyme”.
• biosynthetic pathway also referred to as “metabolic pathway” refers to a set of biochemical reactions, catalyzed by biosynthetic enzymes, which convert one chemical species into another.
• fatty acid biosynthetic pathway (or more simply, “fatty acid pathway”) refers to a set of biochemical reactions that produces fatty acid derivatives (e.g., fatty acids, fatty esters, fatty aldehydes, fatty alcohols, alkanes, alkenes, ketones, and so forth).
• the fatty acid pathway includes fatty acid pathway biosynthetic enzymes (i.e., "fatty acid pathway enzymes”) that can be engineered, as described herein, to produce fatty acid derivatives, and in some embodiments can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics.
• fatty acid pathway biosynthetic enzymes i.e., "fatty acid pathway enzymes”
• a "branched chain fatty acid biosynthetic pathway” i.e., a "BCFA pathway” as described herein includes enzymes sufficient to produce branched fatty acid derivatives.
• recombinant microbial cell refers to a microbial cell (i.e., a
• microorganism that has been genetically modified (i.e., "engineered") by the introduction of genetic material into a "parental microbial cell” (i.e., a host cell) of choice, thereby modifying or altering the cellular physiology and biochemistry of the parental cell.
• a parental microbial cell i.e., a host cell
• the recombinant microbial cell acquires a new or improved property compared to that of the parental microbial cell, such as, for example, the ability to produce a new, or greater quantities of, an intracellular metabolite.
• Recombinant microbial cells provided herein can express a plurality of biosynthetic enzymes (e.g., fatty acid pathway enzymes, such as BCFA pathway enzymes) involved in pathways for the production of, e.g., a branched acyl-CoA, a branched acyl-ACP, or a branched fatty acid derivative (such as a branched fatty acid, branched fatty ester, branched wax ester, branched fatty aldehyde, branched fatty alcohol, branched alkane, branched alkene, branched terminal olefin, branched internal olefin, or branched ketone), from a suitable carbon source.
• biosynthetic enzymes e.g., fatty acid pathway enzymes, such as BCFA pathway enzymes
• a branched acyl-CoA e.g., branched acyl-CoA
• the genetic material introduced into the parental microbial cell contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway (that is, biosynthetic enzymes) for the production of a branched fatty acid derivative, and may also include additional elements for the expression and/or regulation of expression of these genes, such as promoter sequences. Accordingly, recombinant microbial cells described herein have been genetically engineered to express or overexpress biosynthetic enzymes involved in branched chain fatty acid (BCFA) biosynthetic pathways as described herein.
• BCFA branched chain fatty acid
• recombinant microbial cell and “recombinant microorganism” refer not only to the particular recombinant microbial cell/microorganism, but to the progeny or potential progeny of such a microbial cell.
• a recombinant microbial cell can, alternatively or in addition to comprising genetic material introduced into the parental microbial cell, include a reduction, disruption, deletion or a "knocking-out" of a gene or polynucleotide to alter the cellular physiology and biochemistry of the parental microbial cell.
• the recombinant microbial cell acquires a new or improved property (such as, for example, the ability to produce a new or greater quantities of an intracellular metabolite, the ability to improve the flux of a metabolite through a desired pathway, and/or the ability to reduce the production of an undesirable by-product) compared to that of the parental microbial cell.
• a new or improved property such as, for example, the ability to produce a new or greater quantities of an intracellular metabolite, the ability to improve the flux of a metabolite through a desired pathway, and/or the ability to reduce the production of an undesirable by-product
• Branched chain fatty acids are normally produced in bacteria such as Bacillus, Stenotrophomonas, Streptomyces, Listeria, Staphylococcus, and Streptococcus (Kaneda, Microbiol. Rev. 55: 288-302 (1991).
• Branched acyl-CoA molecules are synthesized in such microorganisms by the action of a branched alpha-keto acid dehydrogenase (BKD) complex (Cropp et al., Can J Microbiol 46: 506-14 (2000)). BKD complexes also occur in
• microorganisms such as Pseudomonas that are capable of metabolizing branched-chain amino acids (leucine, isoleucine or valine) or branched-chain a-keto acids as carbon sources (Sokatch et al., J. Bacteriol. 148: 647-652 (1981)).
• Enzymes with beta-ketoacyl ACP synthase III activity that utilize branched-CoA substrates then catalyze the initial condensation of the branched acyl-CoA with malonyl-ACP to form a branched ⁇ -keto acyl- ACP intermediate, which then enters the fatty acid synthase (FAS) cycle to elongate the branched acyl chains.
• FAS fatty acid synthase
• native E. coli lacks components of a BKD complex, and the native E. coli beta-ketoacyl ACP synthase (FabH) enzyme only accepts straight-chain acyl-CoA molecules in the condensation with malonyl-ACP, producing straight-chain ⁇ -keto acyl-ACP intermediates and generating straight-chain fatty acids.
• FSH beta-ketoacyl ACP synthase
• the invention is based in part on the discovery that by engineering microorganisms to introduce or to improve biosynthetic pathways involving the generation of branched chain alpha-keto acids and branched chain acyl-CoA intermediates from simple sugars, metabolic flux through branched chain pathway intermediates is created or is enhanced, and compositions of branched fatty acid products are optimized, resulting in efficient microbial production of branched chain fatty acids and branched chain fatty acid derivatives from simple sugars or biomass.
• Pathways that direct metabolic flux from a starting material, such as a sugar, to a branched acyl-CoA intermediate, to a branched acyl-ACP intermediate, and to a branched fatty acid product or branched fatty acid derivative product, can be engineered in an industrially useful microorganism.
• the invention includes a recombinant microbial cell comprising polynucleotides encoding one or more enzymes which participate in the biosynthesis of a branched acyl-ACP intermediate when the microorganism is cultured in the presence of a carbon source under conditions effective to expresses the polynucleotides.
• the recombinant microbial cell further comprises one or more
• polynucleotides each which encodes a polypeptide having a fatty acid derivative enzyme activity, wherein the recombinant microbial cell produces a branch fatty acid derivative when cultured in the presence of a carbon source under conditions sufficient to expresses the polynucleotides.
• the invention also includes methods of making branched fatty acid derivatives comprising culturing a recombinant microbial cell of the invention.
• the recombinant microbial cell can be a filamentous fungi, an algae, a yeast, or a prokaryote such as a bacterium (e.g., an E. coll or a Bacillus sp).
• branched fatty acid derivatives such as, branched fatty acids, branched fatty esters (including branched fatty acid methyl esters (branched-FAMEs), branched fatty acid ethyl esters (branched-FAEEs), and branched wax esters), branched fatty aldehydes, branched fatty alcohols, branched ketones, and branched hydrocarbons (including branched alkanes, branched alkenes, branched terminal olefins, branched internal olefins)) can be produced in a recombinant microbial cell of the invention via the branched fatty acid biosynthetic pathway ("BCFA pathway”) depicted in the
• the recombinant microbial cell utilizes a branched acyl-CoA molecule as a "primer" for the initiation of the branched fatty acyl chain elongation process.
• the branched fatty acyl elongation process initially involves
• the branched ⁇ -ketoacyl-ACP intermediate undergoes keto-reduction, dehydration and enoyl-reduction at the ⁇ -carbon to form an initial branched acyl-ACP intermediate, which undergoes further cycles of condensation with malonyl-ACP, keto-reduction, dehydration, and enoyl-reduction to form branched acyl-ACP intermediates of increasing length.
• the elongated branched acyl-ACP intermediate is then converted to a branched fatty acid derivative (such as, a branched fatty acid, a branched fatty ester, a branched fatty aldehyde, a branched fatty alcohol, a branched hydrocarbon, or a branched ketone).
• a branched fatty acid derivative such as, a branched fatty acid, a branched fatty ester, a branched fatty aldehyde, a branched fatty alcohol, a branched hydrocarbon, or a branched ketone.
• the straight- chain primer molecule acetyl-CoA initially condenses with a malonyl-ACP molecule to form a straight-chain ⁇ -keto acyl-ACP intermediate, which likewise undergoes cycles of keto- reduction, dehydration, enoyl-reduction and condensation with additional malonyl-ACP molecules, to ultimately produce, e.g., a straight-chain fatty acid.
• the above-noted branched acyl-CoA "primer" molecule can be supplied to the BCFA biosynthetic pathway of the recombinant microbial cell of the invention by a number of methods, as follows.
• a branched acyl-CoA molecule is generated by the native biosynthetic machinery of the microbial cell (e.g., is endogenous to the parental microbial cell).
• one or more enzymes endogenous to the parental microbial cell which contribute to the production of branched acyl-CoA can be overexpressed in the recombinant microbial cell.
• a branched acyl-CoA molecule is produced in the recombinant microbial cell by engineering the cell to express exogenous enzymes, such as one or more components of an exogenous BKD complex, which diverts metabolic flux through branched a-keto acid intermediates to produce branched acyl-CoA molecules, as represented by step (C) of Figure 1.
• exogenous enzymes such as one or more components of an exogenous BKD complex
• Polynucleotides encoding components of the BKD complex can be obtained from
• microorganisms that normally produce branched chain fatty acids or can metabolize branched-chain amino acids or branched a-ketoacids, including, but not limited to, strains of Bacillus, Pseudomonas, Streptomyces, Listeria, Staphylococcus, and Streptococcus.
• a BKD complex comprises three components: an El component having a-keto acid dehydrogenase activity (e.g., EC 1.2.4.4), which, depending on the source, may be a single polypeptide (that is, a monomer), or, two different polypeptides (i.e., a heterodimer) denoted Elalpha and Elbeta; an E2 component having lipoamide acyltransferase activity (e.g., EC 2.3.1.168), and a third component, denoted E3, having dihydrolipoamide dehydrogenase activity (e.g., EC 1.8.1.4).
• Both the El (or Elalpha/Elbeta) and E2 components of the BKD complex utilize branched substrates.
• an enzyme having dihydrolipoamide dehydrogenase activity e.g., EC 1.8.1.4
• a microbial cell which does not normally produce branched fatty acids (such as E. coli) but which can nevertheless utilize branched chain substrates
• BKD E3 component derived from a strain that normally produces branched chain fatty acids or metabolizes branched-chain amino acids or branched a-ketoacids .
• one or more polynucleotide sequences each encoding a polypeptide having a BKD activity (a-keto acid acid dehydrogenase activity, lipoamide acyltransferase activity, or dihydrolipoamide dehydrogenase activity ) which is endogenous to the parental microbial cell is overexpressed in the recombinant microbial cell.
• one or more polynucleotide sequences each encoding a polypeptide having a BKD activity (a-keto acid acid dehydrogenase activity, lipoamide acyltransferase activity, or dihydrolipoamide dehydrogenase activity ) which is exogenous to the parental microbial cell is expressed or overexpressed in the recombinant microbial cell.
• Polynucleotide sequences encoding polypeptides having BKD activities can be obtained from a microorganism that normally produces branched chain fatty acids or can metabolize branched-chain amino acids or branched a-ketoacids (including, but not limited to, strains of Bacillus, Pseudomonas, Streptomyces, Listeria, Staphylococcus, and Streptococcus).
• the polynucleotide sequence is modified to generate a variant polypeptide having a BDK activity and an improved property, compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• BKD component polypeptides and nucleic acids encoding such polypeptides for use in engineering part (C) of the BCFA biosynthetic pathway are provided in Table 4, below.
• a branched acyl-CoA molecule is produced in the recombinant microbial cell by engineering the cell to express or overexpress certain transport/activation enzymes that participate in the conversion of a branched short-chain carboxylic acid substrate to the branched acyl-CoA molecule.
• the transport/activation enzymes can include, without limitation, a phosphotransbutyrylase (ligase) (e.g., EC
• the recombinant microbial ceil expresses a phosphotransbutyrylase (ligase) and a butyrate kinase from Clostridium acetobutylicum.
• the branched short-chain carboxylic acid substrate such as isobutyrate, isovalerate, or 2-methyl-butyrate
• the branched acyl-CoA molecule such as, isobutyryl-CoA, isovaleryl-CoA, or 2-methyl-butyryl-CoA).
• such a recombinant microbial cell is cultured in the presence of (i.e., is "fed") the branched short-chain carboxylic acid substrate, e.g., isobutyrate, isovalerate, or 2-methyl- butyrate, resulting in the production of isobutyryl-CoA, isovaleryl-CoA, or 2-methyl-butyryl- CoA, respectively.
• the branched short-chain carboxylic acid substrate e.g., isobutyrate, isovalerate, or 2-methyl- butyrate
• the branched acyl-CoA molecule isobutyryl-CoA is produced in the recombinant microbial cell by engineering the cell to express a crotonyl-CoA reductase (Ccr, EC 1.6.5.5, 1.1.1.1) and an isobutyryl-CoA mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds, ! Bacteriol. 179:5157, 1997).
• a crotonyl-CoA reductase Ccr, EC 1.6.5.5, 1.1.1.1
• isobutyryl-CoA mutase large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2
• Non-limiting examples of ccr and icm genes include the ccr, icmA and icmB genes from Streptomyces coelicolor (e.g., NCBI Accession Numbers NP_630556, NP_629554, and NP_630904, respectively), and the ccr, icmA and icmB genes from Streptomyces
• cinnamonensis e.g., NCBI accession numbers AAD53915, AAC08713, and AJ246005, respectively.
• a branched acyl-CoA molecule is produced in the recombinant microbial cell by engineering the cell to express enzymes that direct metabolic flux from simple starting materials (e.g., sugars, such as glucose) to generate branched a- keto acid intermediates, which are then acted upon by the native biosynthetic machinery of the particular recombinant microbial cell, or, by BKD complex components engineered in the recombinant microbial cell (for example, as described above), to generate branched acyl-CoA molecules.
• simple starting materials e.g., sugars, such as glucose
• BKD complex components engineered in the recombinant microbial cell for example, as described above
• the branched acyl-CoA molecule serves as a primer for branched acyl-chain elongation. Initiation of the elongation process involves condensation of the branched acyl-CoA with a malonyl-ACP molecule to form a branched ⁇ -ketoacyl-ACP intermediate.
• This step is catalyzed in the recombinant microbial cell by an enzyme having ⁇ -ketoacyl-ACP synthase III activity (e.g., EC 2.3.1.180) which utilizes a branched acyl-CoA molecule as a substrate (in other words, an enzyme having "branched chain ⁇ -ketoacyl-ACP synthase III" activity).
• the enzyme can be endogenous to the recombinant microbial cell (for example, if the parental microbial cell normally produces branched chain fatty acids), or exogenous the recombinant microbial cell.
• a polynucleotide encoding a polypeptide endogenous to the parental microbial cell having branched chain ⁇ -ketoacyl-ACP synthase III activity is overexpressed in the recombinant microbial cell.
• a polynucleotide encoding a polypeptide having branched chain ⁇ -ketoacyl-ACP synthase III activity which is exogenous to the parental microbial cell is expressed or overexpressed in the recombinant microbial cell.
• a polynucleotide sequence encoding a polypeptide having branched chain ⁇ - ketoacyl-ACP synthase III activity can be obtained from a microbial cell that normally produces branched chain fatty acids (including, but not limited to, strains of Bacillus, Streptomyces, Listeria, Staphylococcus, and Streptococcus).
• the polynucleotide sequence is modified to generate a variant polypeptide having branched chain ⁇ -ketoacyl-ACP synthase III activity and an improved property, compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media
• Non-limiting examples of ⁇ -ketoacyl-ACP synthase III enzymes and genes encoding such enzymes for use in engineering part (D) of the branched fatty acid pathway are provided in Table 5, below.
• One or more enzymes endogenous to the parental microbial cell may compete for substrate with enzymes of the engineered BCFA biosynthetic pathway in the recombinant microbial cell, or may break down or otherwise divert an intermediate in the BCFA biosynthetic pathway; genes encoding such undesired endogenous enzymes may be attenuated to increase the production of branched fatty acid derivatives by the recombinant microbial cell.
• E. colifabH (UniProtKB/Swiss-Prot Protein Accession Number P0A6R0), encoded by the E. colifabH gene, primarily utilizes short straight-chain acyl-CoA molecules such as acetyl-CoA, but does not utilize branched acyl-CoA molecules, and thus competes with enzymes of the branched chain pathway for malonyl-ACP and other substrates and diverts metabolic flux away from the BCFA pathway. Deleting or otherwise reducing the expression of the E. colifabH gene thus directs biosynthesis in recombinant E. coli away from straight-chain and more towards branched ⁇ -ketoacyl-ACP intermediates, and ultimately more towards branched-chain fatty acid production .
• the branched ⁇ -ketoacyl-ACP intermediate generated in part (D) of the BCFA pathway can undergo elongation by successive cycles of keto-reduction, dehydration and enoyl-reduction at the beta carbon and further condensation with malonyl- ACP molecules catalyzed by a fatty acid synthase (FAS) complex, such as for example a Type II FAS complex, adding 2-carbon units to the lengthening chain of the branched acyl-ACP intermediate as represented by part (E) of Figure 1.
• FES fatty acid synthase
• an endogenous FAS complex native to the recombinant microbial cell catalyzes cycles of condensation with malonyl-ACP / keto-reduction / dehydration / enoyl-reduction to produce the branched acyl-ACP intermediate.
• Branched fatty acid derivatives (such as branched fatty acids, branched fatty esters, branched fatty aldehydes, branched fatty alcohols, branched hydrocarbons, and branched ketones) can be produced from the branched acyl-ACP intermediate, as will be described in more detail below.
• the recombinant microbial cell further comprises one or more polynucleotide sequences each encoding a polypeptide having fatty acid derivative enzyme activity, such as thioesterase (e.g., TesA), decarboxylase, carboxylic acid reductase (CAR; e.g., CarB), alcohol dehydrogenase / aldehyde reductase; aldehyde decarbonylase, fatty alcohol forming acyl-CoA reductase (FAR), acyl ACP reductase (AAR), ester synthase, or acyl-CoA reductase (ACR1), OleA, OleCD, or OleBCD, wherein the microbial cell produces a composition comprising a branched fatty acid, a branched fatty ester (such as a branched fatty methyl ester, branched fatty ethyl ester,
• thioesterase
• the invention also includes methods for the production of a branched fatty acid derivative comprising culturing a recombinant microbial cell of the invention.
• the branching of the fatty acid derivative molecules can occur in the iso- configuration or the anteiso- configuration.
• Branched chain fatty acids and their derivatives, particularly in the anteiso-configuration, are preferred components of fuel compositions, due to lower melting points and higher oxidative stabilities compared to the non-branched (i.e., straight-chain) compounds.
• the recombinant microbial cell can be modified to generate a particular branched acyl-CoA molecule, which serves as a primer for the initiation of chain elongation.
• the structure of the branched acyl-CoA primer molecule in part determines the structure of the final product.
• condensation of the branched acyl-CoA molecule 2-methyl-butyryl-CoA with malonyl-ACP catalyzed by a branched chain ⁇ -ketoacyl-ACP synthase III generates the anteiso-branched ⁇ -ketoacyl-ACP intermediate 4-methyl-3-oxo-hexanoyl-ACP, leading to production of anteiso-branched acyl-ACP intermediates and ultimately anteiso-branched fatty acid derivatives ( Figure 2A).
• the present invention is based in part on the discovery that directing the flux of certain amino acid biosynthetic metabolites to the production of branched a-keto acid intermediates, and diverting those branched a-keto acid intermediates to conversion into branched acyl-CoAs and entry into the fatty acid biosynthetic pathway optimizes the structures and improve the yields of branched chain fatty acid products.
• the invention includes a recombinant microbial cell comprising polynucleotides encoding one or more enzymes (i.e., "BCFA pathway enzymes") which participate in the conversion of a sugar to the branched a-keto acid molecule 2-keto- 3-methylvalerate (also known as a-keto-3-methylvalerate) when the microorganism is cultured in the presence of a carbon source under conditions sufficient to expresses the polynucleotides.
• the 2-keto-3-methylvalerate molecule is a branched a-keto acid intermediate in the microbial production of anteiso-branched fatty acid derivatives according to the BCFA pathway (see Figure 2A and Figure3B).
• the invention includes a recombinant microbial cell comprising polynucleotides encoding one or more BCFA pathway enzymes which participate in the biosynthesis of an anteiso-branched acyl-ACP intermediate when the microbial cell is cultured in the presence of a carbon source under conditions sufficient to expresses the polynucleotides.
• the recombinant microbial cell further comprises one or more polynucleotides encoding one or more polypeptides each having a fatty acid derivative enzyme activity, wherein the recombinant microbial cell produces an anteiso- branched fatty acid derivative when cultured in the presence of a carbon source under conditions sufficient to expresses the polynucleotides.
• the invention includes methods for the production of
• compositions comprising anteiso-branched fatty acid derivatives, comprising culturing a recombinant microbial cell of the invention.
• Figures 3A and 3B show exemplary biosynthetic pathways for the conversion of a starting material (e.g., a sugar, such as glucose) to an anteiso-branched a-keto acid intermediate, 2-keto-3-methylvalerate, which is then converted to an anteiso-branched acyl-CoA primer, 2-methyl-butyryl-CoA.
• a starting material e.g., a sugar, such as glucose
• a starting material e.g., a sugar, such as glucose
• 2-keto-3-methylvalerate anteiso-branched acyl-CoA primer
• Condensation of the 2-methyl-butyryl-CoA primer with malonyl-ACP results in an anteiso-branched ⁇ -keto acyl-ACP intermediate, 4-methyl-3- oxo-hexanoyl-ACP.
• the anteiso-branched ⁇ -keto acyl-ACP intermediate is a starter unit for further cycles of FAS-catalyzed elongation by condensation with additional malonyl-ACP molecules to generate anteiso-branched acyl-ACP, according to the general pathway diagrammed in Figure 1.
• the anteiso-branched acyl-ACP is then converted in further biocatalytic steps, catalyzed by one or more fatty acid derivative enzymes, to produce an anteiso-branched fatty acid derivative.
• the first pathway leading to the common a-ketobutyrate intermediate involves production of the pathway intermediate threonine by threonine biosynthetic enzymes, followed by the deamination of threonine to ⁇ -ketobutyrate catalyzed by an enzyme with threonine dehydratase activity.
• increasing metabolic flux to the pathway intermediate threonine can be accomplished by expressing polynucleotides encoding enzymes involved in threonine biosynthesis, including enzymes with aspartate kinase activity (e.g., EC 2.7.2.4; also termed aspartokinase activity), which catalyzes the conversion of aspartate to aspartyl phosphate; aspartate-semialdehyde dehydrogenase activity (e.g., EC 1.2.1.11), which catalyzes the conversion of aspartyl phosphate to aspartate semialdehyde; homoserine dehydrogenase activity (e.g., EC 1.1.1.3), which catalyzes the conversion of aspartate semialdehyde to homoserine; homoserine kinase activity (e.g., EC 2.7.1.39), which catalyzes the conversion of homoserine to O-phospho-
• the recombinant microbial cell is engineered to recombinantly express one or more polynucleotides selected from: a polynucleotide encoding a polypeptide having aspartate kinase activity, wherein the polypeptide catalyzes the conversion of aspartate to aspartyl phosphate; a
• polynucleotide encoding a polypeptide having aspartate-semialdehyde dehydrogenase activity, wherein the polypeptide catalyzes the conversion of aspartyl phosphate to aspartate semialdehyde; a polynucleotide encoding a polypeptide having homoserine dehydrogenase activity, wherein the polypeptide catalyzes the conversion of aspartate semialdehyde to homoserine; a polynucleotide encoding a polypeptide having homoserine kinase activity, wherein the polypeptide catalyzes the conversion of homoserine to 0- phospho-L-homoserine; a polynucleotide encoding a polypeptide having threonine synthase activity, wherein the polypeptide catalyzes the conversion of O-phospho-L-homoserine to threonine; wherein the recombin
• the polypeptide encoded by recombinantly expressed polynucleotide is present in the recombinant microbial cell at a greater concentration compared to its concentration in the parent microbial cell when cultured under the same conditions, i.e., the polypeptide is "overexpressed" in the recombinant cell.
• the recombinantly expressed polynucleotide can be operatively linked to a promoter which expresses the polynucleotide in the recombinant microbial cell at a greater concentration than is normally expressed in the parental microbial cell when cultured under the same conditions.
• an E is an enzyme that catases the polypeptide encoded by recombinantly expressed polynucleotide.
• coli thrA gene which encodes a Afunctional ThrA with aspartate kinase and homoserine dehydrogenase activities.
• a mutant E. coli thrA gene is used, encoding a variant enzyme with aspartate kinase and homoserine dehydrogenase activities and with reduced feedback inhibition relative to the parent ThrA enzyme
• ThrA* Ogawa-Miyata,Y., et al., Biosci. Biotechnol. Biochem. 65:1149-1154 (2001); Lee J.-H., et al., J. Bacteriol. 185: 5442-5451 (2003)).
• Threonine can be deaminated to a-ketobutyrate (also known as 2-ketobutyrate, 2- oxobutanoate and 2-oxobutyrate) by an enzyme with threonine deaminase activity (e.g., EC 4.3.1.19; also known as threonine ammonia-lyase activity, and was previously classified as EC 4.2.1.16, threonine dehydratase), which catalyzes the conversion of threonine to a- ketobutyrate.
• threonine deaminase activity e.g., EC 4.3.1.19; also known as threonine ammonia-lyase activity, and was previously classified as EC 4.2.1.16, threonine dehydratase
• threonine deaminase activity already present in (i.e., endogenous to) the parental microbial cell is sufficient to catalyze the conversion of threonine to a-ketobutyrate.
• the recombinant microbial cell is engineered to recombinantly express a polypeptide having threonine deaminase activity, wherein the polypeptide catalyzes the conversion of threonine to ⁇ -ketobutyrate.
• the polypeptide having threonine deaminase activity is overexpressed in the recombinant microbial cell.
• BCFA pathway enzymes and polynucleotides encoding such enzymes for use in part (A. l) of the branched fatty acid pathway are provided in Table 1 .
• B. subtil is 168 thrC P04990 NP_391105 142,143
• threonine deaminase threonine ammonia-lyase; previously termed threonine
• Additional polypeptides can be identified, for example, by searching a relevant database (such as the KEGG database (University of Tokyo), the PROTEIN or the GEN E databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)), all which are available on the World Wide Web, for polypeptides categorized by the above noted EC numbers.
• a relevant database such as the KEGG database (University of Tokyo), the PROTEIN or the GEN E databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)
• additional aspartokinase polypeptides can be identified by searching for polypeptides categorized under EC 2.7.2.4; additional homoserine dehydrogenase polypeptides can be identified by searching for polypeptides categorized under EC 1.1.1.3; additional homoserine kinase polypeptides can be identified by searching for polypeptides categorized under EC 2.7.1.39; additional threonine synthase polypeptides can be identified by searching for polypeptides categorized under EC 4.2.3.1; and additional threonine deaminase polypeptides can be identified by searching for polypeptides categorized under EC 4.3.1.19.
• a polynucleotide encoding a parent fatty acid pathway polypeptide (such as a polypeptide described in Table 1 or identified by EC number or by homology to an exemplary polypeptide) is modified using methods well known in the art to generate a variant polypeptide having an enzymatic activity noted above (e.g.,
• aspartokinase activity homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, threonine deaminase activity
• an improved property compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• the second pathway leading to the common 2-ketobutyrate intermediate involves the production of the pathway
• citramalate which is also known as 2-methylmalate
• an enzyme with citramalate synthase activity via an enzyme with citramalate synthase activity, and the conversion of citramalate to 2-ketobutyrate by the action of enzymes with isopropylmalate isomerase and alcohol dehydrogenase activities.
• Citramalate synthase activity (e.g., EC 2.3.1.182), which catalyzes the reaction of acetyl-CoA and pyruvate to form (R)-citramalate, can be supplied by expression of a cimA gene from a bacterium such as Methanococcus jannaschi or Leptospira interrogans (Howell, D.M. et ai., J. Bacteriol. 181(l):331-3 (1999); Xu, H., et al J. Bacteriol. 186:5400-5409(2004)) which encodes a CimA polypeptide such as CimA from M. jannaschii (SEQ ID NO: 156) or L.
• a cimA gene from a bacterium such as Methanococcus jannaschi or Leptospira interrogans (Howell, D.M. et ai., J. Bacteriol. 181(l
• a modified cimA nucleic acid sequence encoding a CimA variant with improved catalytic activity or stability in the recombinant microbial cell and/or reduced feedback inhibition can be used, such as, for example, a CimA variant described by Atsumi S. and Liao J.C. ⁇ Appl. Environ. Microbiol. 74(24): 7802-7808 (2008)), preferably the CimA3.7 variant (SEQ ID NO:158) encoded by the cimA3.7 gene (SEQ ID NO:159).
• a Leptospira interrogans CimA variant (SEQ ID NO:162) can be used.
• Isopropylmalate isomerase activity (EC 4.2.1.33; also termed isopropylmalate dehydratase), which catalyzes the conversion of (R)-citramalate first to citraconate and then to beta- methyl-D-malate, can be provided, for example, by expression of a heterodimeric protein encoded by E. coli or B. subtilis leuCD genes.
• Alcohol dehydrogenase activity EC 1.1.1.85; beta-isopropyl malate dehydrogenase
• beta-methyl-D- malate to 2-ketobutyrate i.e., alpha-keto butyrate
• Non-limiting examples of fatty acid pathway enzymes and polynucleotides encoding such enzymes for use in engineering part (A.2) of the branched fatty acid pathway are provided in Table 2.
• Additional polypeptides can be identified, for example, by searching a relevant database (such as the KEGG database (University of Tokyo), the PROTEIN or the GEN E databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)), all which are available on the World Wide Web, for polypeptides categorized by the above noted EC numbers.
• a relevant database such as the KEGG database (University of Tokyo), the PROTEIN or the GEN E databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)
• additional (R)-citramalate synthase polypeptides can be identified by searching for polypeptides categorized under EC 2.3.1.182; additional isopropyl malate isomerase polypeptides can be identified by searching for polypeptides categorized under EC 4.2.1.33; and additional beta-isopropyl malate dehydrogenase polypeptides can be identified by searching for polypeptides categorized under EC 1.1.1.85.
• a polynucleotide encoding a parent fatty acid pathway polypeptide (such as a polypeptide described in Table 2 or identified by EC number or by homology to an exemplary polypeptide) is modified using methods well known in the art to generate a variant polypeptide having an enzymatic activity noted above (e.g., (R)- citramalate synthase activity, isopropyl malate isomerase activity, beta-isopropyl malate dehydrogenase activity) and an improved property, compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• an enzymatic activity e.g., (R)- citramalate synthase activity
• Pathway Part B 2-Ketobutyrate to 2-Keto-3-Methylvalerate
• the ot-ketobutyrate produced by the first and/or the second pathway can then be converted to the branched ct-keto acid, 2-keto-3-methylvalerate, by the action of enzymes with acetohydroxyacid synthase activity (such as, an AHAS complex), acetohydroxyacid isomeroreductase activity, and dihydroxy acid dehydratase activity, as represented by part (B) of Figure 3B.
• acetohydroxyacid synthase activity such as, an AHAS complex
• acetohydroxyacid isomeroreductase activity acetohydroxyacid isomeroreductase activity
• dihydroxy acid dehydratase activity as represented by part (B) of Figure 3B.
• AHAS acetohydroxyacid synthase
• coli and various other bacteria contain AHAS isozymes designated AHAS I (e.g., encoded by ilvBN genes), AHAS II (e.g., encoded by ilvGM genes) and AHAS III (e.g., encoded by ilvlH genes).
• AHAS I e.g., encoded by ilvBN genes
• AHAS II e.g., encoded by ilvGM genes
• AHAS III e.g., encoded by ilvlH genes.
• the acetohydroxyacid synthase activity present in the parental microbial cell is sufficient to catalyze the reaction of 2-ketobutyrate and pyruvate to 2-aceto-2-hydroxybutyrate.
• the recombinant microbial cell is engineered to recombinantly express AHAS polypeptides having acetohydroxyacid synthase activity, wherein the AHAS polypeptides catalyze the reaction of
• polypeptides having acetohydroxyacid synthase activity are overexpressed in the recombinant microbial cell. If the microbial cell being engineered is an E. coli K-12 strain, and, if E. coli AHAS II activity is desired, an ilvG gene (or an ilvGM gene cluster) must be introduced from a different strain of E. coli, or, the endogenous ilvG gene must be repaired by recombinant methods, since the ilvG gene endogenous to E. coli K-12 is inactive.
• AHAS I and AHAS III activities present in the parental E. coli K-12 cell e.g., encoded by endogenous ilvBN and/or ilvlH genes
• ilvBN and/or ilvlH genes could be utilized.
• genes endogenous to the parental microbial cell that is, non-recombinant native host genes
• native enzymes encoded by those endogenous genes could be utilized for the various steps of part (B) of the pathway.
• Non-limiting examples of fatty acid pathway enzymes and polynucleotides encoding such enzymes suitable for use in part (B) of the branched fatty acid pathway are provided in Table 3.
• E. coli K-12 (llvl) P00893 (1) YP_025294.2 186, 187 MG1655 ilvlH (llvH) P00894 (H) NP_414620 188,189
• B. subtil is 168 ilvC P37253 NP_390707 196, 197
• Additional polypeptides can be identified, for example, by searching a relevant database (such as the KEGG database (University of Tokyo), the PROTEIN or the GENE databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)), all which are available on the World Wide Web, for polypeptides categorized by the above noted EC numbers.
• a relevant database such as the KEGG database (University of Tokyo), the PROTEIN or the GENE databases (Entrez databases; NCBI), the UNIPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)
• isomeroreductase polypeptides can be identified by searching for polypeptides categorized under EC 1.1.1.86; and additional dihydroxyacid dehydratase polypeptides can be identified by searching for polypeptides categorized under EC 4.2.1.9.
• a polynucleotide encoding a parent fatty acid pathway polypeptide (such as a polypeptide described in Table 3 or identified by EC number or by homology to an exemplary polypeptide) is modified using methods well known in the art to generate a variant polypeptide having an enzymatic activity noted above (e.g.,
• acetohydroxyacid synthase activity acetohydroxyacid isomeroreductase activity, dihydroxyacid dehydratase activity
• an improved property compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• Pathway Part C Branched Alpha-Keto Acid to Branched Acyl-CoA
• the branched acyl-CoA molecule is generated from the branched ct-keto acid, as represented by part (C) of Figures 1 and 3B, by the action of a multi-component branched chain alpha-keto acid dehydrogenase (BKD) complex.
• BKD branched chain alpha-keto acid dehydrogenase
• components of the BKD complex can be obtained from a microbial cell that normally produces branched chain fatty acids or can metabolize branched amino acids or branched a- ketoacids (including, but not limited to, strains of Bacillus, Pseudomonas, Streptomyces, Listeria, Staphylococcus, and Streptococcus).
• the BKD complex comprises at least two components: an El component having alpha-keto acid dehydrogenase activity (e.g., EC 1.2.4.4), and which, depending on the source, may be a single polypeptide (that is, a monomer), or, a heterodimer denoted Elalpha and Elbeta; and a E2 component having lipoamide acyltransferase activity (e.g., EC 2.3.1.168). Both the El (or Elalpha/Elbeta) and E2 components utilize branched substrates.
• an El component having alpha-keto acid dehydrogenase activity e.g., EC 1.2.4.4
• E2 component having lipoamide acyltransferase activity e.g., EC 2.3.1.168
• the BDK complex comprises a third component, denoted E3, having dihydrolipoamide dehydrogenase activity (e.g., EC 1.8.1.4); in some instances, an enzyme having dihydrolipoamide dehydrogenase activity and which utilizes branched chain substrates can be obtained from a microbial cell which does not normally produce branched fatty acids (such as E. coli), which may be used in place of a BKD E3 component.
• E3 dihydrolipoamide dehydrogenase activity
• dehydrogenase activity (El activity, e.g., EC 1.2.4.4) and the lipoamide acyltransferase activity (E2 activity, e.g., EC 2.3.1.168) of the BKD complex
• El activity e.g., EC 1.2.4.4
• E2 activity e.g., EC 2.3.1.168
• BKD El or Elalpha/beta
• E2 component polypeptides from microorganisms that normally produce branched fatty acids or can metabolize branched amino acids or branched a-ketoacids, such as, for example, Bacillus subtilis, Pseudomonas putida, Listeria monocytogenes, Micrococcus luteus, and Streptococcus mutans.
• Dihydrolipoamide dehydrogenase activity (E3 activity, e.g., EC 1.8.1.4) can likewise be introduced by expression of a polynucleotide encoding an E3 component from a microorganism that normally produces branched chain fatty acids; alternatively, a polynucleotide encoding a polypeptide with dihydrolipoyl dehydrogenase activity from a microorganism that normally does not produce branched chain fatty acids, but which nevertheless utilizes branched chain substrates (for example, an E.
• E3 activity e.g., EC 1.8.1.4
• coli dihydrolipoyl dehydrogenase can be used, if the recombinant microbial cell being engineered is one that normally produces branched chain fatty acids (and, as such, produces an endogenous BKD complex), one or more endogenous BKD complex components can be overexpressed.
• Non-limiting examples of fatty acid pathway enzymes and polynucleotides encoding such enzymes for use in engineering part (C) of the branched fatty acid pathway are provided in Table 4.
• EC lipoamide acyltransferase dihydrolipoyl transacylase; dihydrolipoyllysine-residue (2-
• Additional polypeptides can be identified, for example, by searching a relevant database (such as the KEGG database (University of Tokyo), the PROTEIN or the GENE databases (Entrez databases; NCBI), the UNIPROTKB or ENZYM E databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)), all which are available on the World Wide Web, for polypeptides categorized by the above noted EC numbers.
• a relevant database such as the KEGG database (University of Tokyo), the PROTEIN or the GENE databases (Entrez databases; NCBI), the UNIPROTKB or ENZYM E databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)
• additional branched chain alpha-keto acid dehydrogenase polypeptides can be identified by searching for polypeptides categorized under EC 1.2.4.4; additional lipoamide acyltransferase polypeptides can be identified by searching for polypeptides categorized under EC 2.3.1.168; and additional dihydrolipoyl dehydrogenase polypeptides can be identified by searching for polypeptides categorized under EC 1.8.1.4.
• BKD complex component polypeptides can also be identified by searching a sequence pattern database, such as the Prosite database (ExPASy Proteomics Server, Swiss Institute of Bioinformatics) for a polypeptide comprising one or more of the sequence motifs listed below. This is readily accomplished, for example, by using the ScanProsite tool which is available on the World Wide Web site of the ExPASy Proteomics Server.
• a branched chain alpha-keto acid dehydrogenase (BKD El-alpha subunit) polypeptide comprises one or more sequence motif selected from:
• amino acid residues in each of the brackets indicate alternative amino acid residues at the particular position
• each x indicates any amino acid residue
• each n in "x(n)" indicates the number of x residues in a contiguous stretch of amino acid residues.
• a branched chain alpha-keto acid dehydrogenase (BKD El- beta subunit) polypeptide comprises one or more sequence motif selected from: V-x-[V,l]-x-G-[ E]-D-V-G-x(2)-G-G-V-F-[R,K]-x-T-x-G-l (SEQ ID NO:36)
• a lipoamide acyltransferase (BKD E2 component) polypeptide comprises one or more sequence motif selected from:
• amino acid residues in each of the brackets indicate alternative amino acid residues at the particular position
• each x indicates any amino acid residue
• each n in "x(n)" indicates the number of x residues in a contiguous stretch of amino acid residues.
• a dihydrolipoyl dehydrogenase (BKD E3 component) polypeptide comprises one or more sequence motif selected from:
• amino acid residues in each of the brackets indicate alternative amino acid residues at the particular position
• each x indicates any amino acid residue
• each n in "x(n)" indicates the number of x residues in a contiguous stretch of amino acid residues.
• a polynucleotide encoding a parent fatty acid pathway polypeptide (such as a BKD complex polypeptide described in Table 4 or identified by EC number or by motif or by homology to an exemplary polypeptide) is modified using methods well known in the art to generate a variant polypeptide having BKD complex enzymatic activity and an improved property, compared to that of the parent polypeptide, which is more suited to the microbial cell and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured; reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• a parent fatty acid pathway polypeptide such as a BKD complex polypeptide described in Table 4 or identified by EC number or by motif or by homology to an exemplary polypeptide
• BKD complex enzymatic activity can be measured according to various known protocols.
• Sokatch et al. described an assay mixture containing: 100 mM potassium phosphate buffer (pH 7.0), 2 mM NAD+, 0.1 mM coenzyme A, 0.2 mM
• the reaction can be initiated by adding 4.0 ⁇ . of the sodium salt of a branched- chain a-keto acid substrate, such as 2-keto-3-methylvalerate (Sokatch et al., J. Bacteriology 148(2):647-652 (1981)).
• a branched- chain a-keto acid substrate such as 2-keto-3-methylvalerate
• the branched acyl-CoA serves as a primer for subsequent FAS- catalyzed elongation steps.
• the initiation of this process involves condensation of the branched acyl-CoA with a malonyl-ACP molecule to form a branched ⁇ -ketoacyl-ACP intermediate.
• the anteiso-branched acyl-CoA molecule 2-methylbutyryl-CoA can condense with malonyl-ACP to form an anteiso-branched ⁇ -ketoacyl-ACP intermediate 4-methyl-3-oxo-hexanoyl-ACP
• the iso-branched acyl-CoA molecule isobutyryl-CoA can condense with malonyl-ACP to form an iso-branched ⁇ -ketoacyl-ACP intermediate 4-methyl- 3-oxo-pentanoyl-ACP ( Figures 2A and 2B).
• This initiation step is catalyzed in the recombinant microbial cell by an enzyme having beta-ketoacyl-ACP synthase activity (such as, a Type III beta-ketoacyl-ACP synthase (e.g., EC 2.3.1.180)) that utilizes branched acyl-CoA molecules (for example, anteiso-branched acyl-CoA molecules) as substrates.
• an enzyme is also referred to herein as an enzyme having "branched chain beta-ketoacyl-ACP synthase activity".
• a polynucleotide sequence encoding an polypeptide having branched chain beta-ketoacyl-ACP synthase activity can be obtained from a microbial cell that normally produces branched chain fatty acids (including, but not limited to, strains of Bacillus, Streptomyces, , Stenotrophomonas, Listeria, Staphylococcus, and Streptococcus), and expressed or overexpressed in the recombinant microbial cell.
• branched chain fatty acids including, but not limited to, strains of Bacillus, Streptomyces, , Stenotrophomonas, Listeria, Staphylococcus, and Streptococcus
• one or more enzymes endogenous to the parental microbial cell might compete for substrate with enzymes of the engineered branched fatty acid biosynthetic pathway in the recombinant microbial cell, or might break down or otherwise divert an intermediate in the biosynthetic pathway; genes encoding such undesired endogenous enzymes can be attenuated to increase branched fatty acid production in the recombinant microbial cell.
• the endogenous ⁇ -ketoacyl-ACP synthase III (UniProtKB/Swiss-Prot Protein Accession Number P0A6R0), encoded by the E.
• colifabH gene primarily utilizes short straight-chain acyl-CoA molecules such as acetyl-CoA, but does not utilize branched acyl-CoA molecules, and thus competes with enzymes of the branched chain pathway for malonyl-ACP and other substrates and diverts flux away from the BCFA pathway. Deleting or otherwise attenuating the E. colifabH gene thus directs fatty acid biosynthesis in a recombinant E. coli comprising a BCFA pathway more towards the production of branched fatty acids.
• Other endogenous enzymes that may be undesired and which may be attenuated in the recombinant microbial cell include, for example, the E. coli fadE gene encoding an acyl-CoA dehydrogenase which metabolizes acyl-CoA
• Non-limiting examples of fatty acid pathway enzymes and polynucleotides encoding such enzymes for use in engineering part D of the branched fatty acid pathway are provided in Table 5.
• Table 5 Non-limiting examples of enzymes and coding sequences for use in Part D of the BCFA biosynthetic pathways shown in Figures 1 and 3B.
• Additional beta-ketoacyl-ACP III synthase polypeptides can be identified, for example, by searching a relevant database (such as the KEGG database (University of Tokyo), the PROTEIN or the GENE databases (Entrez databases; NCBI), the UN IPROTKB or ENZYME databases (ExPASy; Swiss Institute of Bioinformatics), and the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig)), all which are available on the World Wide Web, for polypeptides categorized under EC 2.3.1.180.
• Additional beta-ketoacyl-ACP synthase III polypeptides can also be identified by searching a sequence pattern database, such as the Prosite database (ExPASy Proteomics Server, Swiss Institute of Bioinformatics) for a polypeptide comprising one or more of the sequence motifs listed below. This is readily accomplished, for example, by using the ScanProsite tool which is available on the World Wide Web site of the ExPASy Proteomics Server.
• a sequence pattern database such as the Prosite database (ExPASy Proteomics Server, Swiss Institute of Bioinformatics) for a polypeptide comprising one or more of the sequence motifs listed below. This is readily accomplished, for example, by using the ScanProsite tool which is available on the World Wide Web site of the ExPASy Proteomics Server.
• a beta-ketoacyl-ACP synthase III polypeptide comprises one or more sequence motif selected from:
• amino acid residues in each of the brackets indicate alternative amino acid residues at the particular position
• each x indicates any amino acid residue
• each n in "x(n)" indicates the number of x residues in a contiguous stretch of amino acid residues.
• a polynucleotide encoding a parent fatty acid pathway polypeptide (such as a polypeptide described in Table 5 or identified by EC number or by motif or by homology to an exemplary polypeptide) is modified using methods well known in the art to generate a variant polypeptide having branched chain beta-ketoacyl-ACP III synthase activity, and an improved property, compared to that of the parent polypeptide, which is more suited to the microorganism and/or to the pathway being engineered; such as, for example, increased catalytic activity or improved stability under conditions in which the recombinant microbial cell is cultured, reduced inhibition (e.g., reduced feedback inhibition) by a cellular metabolite or by a culture media component, and the like.
• a parent fatty acid pathway polypeptide such as a polypeptide described in Table 5 or identified by EC number or by motif or by homology to an exemplary polypeptide
• an improved property compared to that of the parent polypeptide,
• the invention includes an isolated polypeptide comprising a sequence having at least 80% identity to one of SEQ ID NOs:84, 86, 88, 90, 92, 94, 96, 98, 102, 104, 106, and 108, and comprising a substitution at position W310 or at an equivalent position thereto, wherein the polypeptide has beta-ketoacyl-ACP synthase activity.
• the invention also includes an isolated polynucleotide encoding any one of said polypeptides.
• the polypeptide comprises a W310G substitution.
• the polypeptide comprises a sequence having at least 80% identity to SEQ ID NO: 98 and comprises the substitution W310G.
• the polypeptide comprises the sequence SEQ ID NO:100.
• the polynucleotide encodes the sequence SEQ ID NO:100, or comprises the sequence SEQ ID NO:101.
• Enzymatic activity and specificity for branched substrates of beta ketoacyl-ACP synthases can be determined using known methods. For example, Choi er al. (J.
• ⁇ -ketoacyl-ACP synthase (“FabH") activity against acetyl-CoA substrates, which can be modified to use branched-chain acyl-CoA substrates.
• the assay contains 25 ⁇ ACP, 1 mM ⁇ -mercaptoethanol, 65 ⁇ malonyl-CoA, 45 ⁇ [l- 14 C]acetyl-CoA (specificity activity about 45.8 Ci/mol), E.coli FadD (0.2 ⁇ g), and 0.1 M sodium phosphate buffer (pH 7.0) in a final volume of 40 ⁇ 1_.
• [1- 1 C]acetyl-CoA can be substituted with a 1 C labeled branched acyl-CoA.
• the reaction is initiated by the addition of FabH, and the mixture is incubated at 37°C for 12 minutes. A 35 mL aliquot is then removed and deposited on a Whatman 3 MM filter disc. The discs are then washed with three changes (20 mL/disc for 20 minutes each) of ice-cold trichloroacetic acid. The concentration of the trichloroacetic acid is then reduced from 10 to 5 to 1% in each successive wash. The filters are dried an counted in 3 mL of scintillation cocktail.
• FabH activity can be determined using gel electrophoresis to separate and quantitate the products (Choi er al., supra).
• the assay mixture contains 25 ⁇ ACP, 1 mM ⁇ -mercaptoethanol, 70 ⁇ [2- 14 C] malonyl-CoA (specific activity, ⁇ 9 Ci/mol), 45 ⁇ of a CoA-substrate (such as acetyl-CoA; or, to assay branched-chain ⁇ -ketoacyl-ACP synthase, isobutyryl-CoA, isovaleryl-CoA, or 2-methylbutyryl-CoA), FadD (0.2 ⁇ g), 100 ⁇ NADPH, FabG (0.2 ⁇ g) and 0.1 M sodium phosphate buffer (pH 7.0) in a final volume of 40 ⁇ .
• the reaction can be initiated by the addition of FabH.
• the mixture is incubated at 37°C for 12 minutes and then placed in an ice slurry, gel loading buffer is then added, and the mixture is loaded onto a conformationally sensitive 13% polyacrylamide gel containing 0.5 to 2.0 M urea.
• Electrophoresis can be performed at 25°C at 32 mA/gel.
• the gels are then dried, and the bands quantitated by exposure of the gel to a Phospholmager screen. Specific activity can be calculated from the slopes of the plot of product formation vs. FabH protein concentration in the assay.
• Pathway Part E Branched ⁇ -Ketoacyl-ACP to Branched Fatty Acyl-ACP
• the branched ⁇ -ketoacyl-ACP intermediate generated in part (D) can undergo elongation by successive cycles of condensation with malonyl-ACP / keto-reduction / dehydration / enoyl-reduction, catalyzed by a fatty acid synthase (FAS) complex, such as, for example, a type II fatty acid synthase complex, thereby adding 2-carbon units to the lengthening fatty acid chain of the resulting branched acyl-ACP, as represented by part (E) of Figure 1.
• FAS fatty acid synthase
• an anteiso-branched ⁇ -ketoacyl-ACP intermediate produces an anteiso-branched acyl-ACP intermediate.
• an iso-branched ⁇ - ketoacyl-ACP intermediate produces an iso-branched acyl-ACP intermediate.
• a FAS enzyme complex (such as, for example, a Type II FAS complex) endogenous to the microbial cell is used to catalyze cycles of condensation with malonyl- ACP / keto-reduction / dehydration / enoyl-reduction to produce the branched acyl-ACP intermediate.
• Branched fatty acid derivatives can be produced by a recombinant microbial cell of the invention.
• the branched acyl-ACP intermediate is converted to a fatty acid derivative in a reaction catalyzed by an enzyme having fatty acid derivative activity (i.e., a fatty acid derivative enzyme).
• a fatty acid derivative enzyme can, for example, convert a branched acyl-ACP to an initial fatty acid derivative, or, can convert the initial fatty acid derivative to a second fatty acid derivative.
• the initial fatty acid derivative is converted to a second fatty acid derivative by an enzyme having a different fatty acid derivative activity.
• the second fatty acid derivative is further converted to a third fatty acid derivative by another fatty acid derivative enzyme, and so on.
• the recombinant microbial cell further comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having a fatty acid derivative enzyme activity, wherein the recombinant microbial cell produces a branched fatty acid derivative when cultured in the presence of a carbon source under conditions effective to express the polynucleotides.
• the fatty acid derivative activity comprises thioesterase activity, wherein the recombinant microbial cell produces branched fatty acids; ester synthase activity, wherein the recombinant microbial cell produces branched fatty esters; fatty aldehyde biosynthesis activity, wherein the recombinant microbial cell produces branched fatty aldehydes; fatty alcohol biosynthesis activity, wherein the recombinant microbial cell produces branched fatty alcohols; ketone biosynthesis activity, wherein the recombinant microbial cell produces branched ketones; or hydrocarbon biosynthesis activity, wherein the recombinant microbial cell produces branched hydrocarbons.
• the recombinant microbial cell comprises polynucleotides encoding two or more polypeptides, each polypeptide having fatty acid derivative enzyme activity.
• the recombinant microbial cell expresses or overexpresses one or more polypeptides having fatty acid derivative enzyme activity as described hereinabove, wherein the recombinant microbial cell produces a composition comprising branched fatty acids, branched fatty esters, branched wax esters, branched fatty aldehydes, branched fatty alcohols, branched alkanes, branched alkanes branched internal olefins, branched terminal olefins, or branched ketones.
• fatty acid derivative enzymes and fatty acid derivatives produced by reactions catalyzed by such enzymes, in accordance with various embodiments of the invention.
• the recombinant microbial cell comprises a polynucleotide encoding a thioesterase, and the branched fatty acyl-ACP intermediate produced by the recombinant microbial cell is hydrolyzed by the thioesterase (e.g., 3.1.1.5, EC 3.1.2.- ; such as, for example, EC 3.1.2.14) resulting in production of a branched fatty acid.
• a composition comprising branched fatty acids (also referred to herein as a "branched fatty acid composition") is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the polynucleotides.
• the composition is recovered from the cell culture.
• the recombinant microbial cell comprises a polynucleotide encoding a polypeptide having thioesterase activity, and one or more additional polynucleotides encoding polypeptides having other fatty acid derivative enzyme activities.
• the branched fatty acid produced by the action of the thioesterase is converted by one or more enzymes having different fatty acid derivative enzyme activities to another branched fatty acid derivative, such as, for example, a branched fatty ester, a branched fatty aldehyde, a branched fatty alcohol, or a branched hydrocarbon.
• an anteiso-branched fatty acyl-ACP intermediate reacts with a thioesterase to form an anteiso-branched fatty acid.
• the anteiso-branched fatty acid can be recovered from the cell culture, or can be further converted to another anteiso-branched fatty acid derivative, such as an anteiso-branched fatty ester, an anteiso-branched fatty aldehyde, an anteiso-branched fatty alcohol, or an anteiso-branched hydrocarbon.
• the chain length of a fatty acid, or a fatty acid derivative made therefrom can be selected for by modifying the expression of certain thioesterases.
• Thioesterase influences the chain length of fatty acids produced as well as that of the derivatives made therefrom.
• the recombinant microbial cell can be engineered to express, overexpress, have attenuated expression, or not to express one or more selected thioesterases to increase the production of a preferred fatty acid or fatty acid derivative substrate.
• Ci 0 fatty acids can be produced by expressing a thioesterase that has a preference for producing Ci 0 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than Ci 0 fatty acids (e.g., a thioesterase which prefers to produce C i4 fatty acids). This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 10.
• C 14 fatty acids can be produced by attenuating endogenous thioesterases that produce non-Ci 4 fatty acids and expressing thioesterases that use C 14 - ACP.
• C 12 fatty acids can be produced by expressing thioesterases that use C 1 2-ACP and attenuating thioesterases that produce non-Ci 2 fatty acids.
• Fatty acid overproduction can be verified using methods known in the art, for example, by use of radioactive precursors, HPLC, or GC-MS subsequent to cell lysis.
• thioesterases and polynucleotides encoding them for use in the branched fatty acid pathway are provided in Table 6 and in PCT
• the recombinant microbial cell produces a branched fatty ester (e.g., an anteiso-branched fatty ester or an iso-branched fatty ester), such as, for example, a branched fatty acid methyl ester or a branched fatty acid ethyl ester or a branched wax ester.
• a branched fatty acid produced by the recombinant microbial cell is converted into the branched fatty ester .
• the recombinant microbial cell comprises a polynucleotide encoding a polypeptide (i.e., an enzyme) having ester synthase activity (also referred to herein as an "ester synthase polypeptide” or an “ester synthase enzyme”), and the branched fatty ester is produced by a reaction catalyzed by the ester synthase polypeptide expressed or overexpressed in the recombinant microbial cell.
• a polypeptide i.e., an enzyme
• ester synthase activity also referred to herein as an "ester synthase polypeptide” or an "ester synthase enzyme
• a composition comprising branched fatty esters (also referred to herein as a "branched fatty ester composition”), produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the polynucleotides, is recovered from the cell culture.
• the recombinant cell produces a branched fatty ester composition comprising anteiso-branched fatty esters.
• Ester synthase polypeptides include, for example, an ester synthase polypeptide classified as EC 2.3.1.75, or any other polypeptide which catalyzes the conversion of an acyl- thioester to a fatty ester, including, without limitation, a wax-ester synthase, an acyl- CoA:alcohol transacylase, an acyltransferase, or a fatty acyl-CoA:fatty alcohol
• the polynucleotide may encode wax/dgat, a bifunctonal ester synthase/acyl-CoA:diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobocter sp. Strain ADP1, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus.
• the ester synthase polypeptide is an Acinetobocter sp. diacylglycerol O-acyltransferase (wax-dgaT; UniProtKB Q8GGG1, GenBank AA017391) or Simmondsia chinensis wax synthase
• the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant microbial cell.
• the recombinant microbial cell further comprises a
• the recombinant microbial cell produces a branched fatty ester, such as, for example, a branched fatty acid methyl ester or a branched fatty acid ethyl ester, wherein the recombinant microbial cell expresses a polynucleotide encoding an ester synthase / acyltransferase polypeptide classified as 2.3.1.20, such as AtfAl (an
• the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant microbial cell.
• the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase.
• the recombinant microbial cell produces a branched fatty ester, such as, for example, a branched fatty acid methyl ester or a branched fatty acid ethyl ester, wherein the recombinant microbial cell expresses a polynucleotide encoding a ester synthase polypeptide, such as ES9 (a wax ester synthase from Marinobacter
• hydrocarbonoclasticus DSM 8798 UniProtKB A3RE51, GenBank ABO21021, encoded by the ws2 gene), or ES376 (another wax ester synthase derived from Marinobacter
• the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant microbial cell.
• the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase.
• ester synthase polypeptides and polynucleotides encoding them suitable for use in these embodiments include those described in PCT Publication Nos. WO 2007/136762 and WO2008/119082 which are incorporated by reference herein.
• the recombinant microbial cell produces a branched fatty aldehyde.
• a branched fatty acid produced by the recombinant microbial cell is converted into the branched fatty aldehyde.
• the branched fatty aldehyde produced by the recombinant microbial cell is then converted into a branched fatty alcohol or a branched hydrocarbon.
• the recombinant microbial cell comprises a polynucleotide encoding a polypeptide (i.e., an enzyme) having fatty aldehyde biosynthesis activity (also referred to herein as a "fatty aldehyde biosynthesis polypeptide” or a "fatty aldehyde biosynthesis enzyme”), and the branched fatty aldehyde is produced by a reaction catalyzed by the fatty aldehyde biosynthesis polypeptide expressed or overexpressed in the recombinant microbial cell.
• a polypeptide i.e., an enzyme
• fatty aldehyde biosynthesis activity also referred to herein as a "fatty aldehyde biosynthesis polypeptide” or a "fatty aldehyde biosynthesis enzyme”
• a composition comprising branched fatty aldehydes (also referred to herein as a "branched fatty aldehyde composition”), produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the polynucleotides, is recovered from the cell culture.
• the recombinant cell produces a branched fatty aldehyde composition comprising anteiso-branched fatty aldehydes.
• the branched fatty aldehyde is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a polypeptide having a fatty aldehyde biosynthesis activity such as carboxylic acid reductase (CAR) activity (encoded, for example, by a car gene).
• CAR carboxylic acid reductase
• FadD9 EC 6.2.1.-, UniProtKB Q50631, GenBank NP_217106
• CarA GenBank ABK75684
• CarB GenBank YP889972
• the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase.
• the branched fatty aldehyde is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide, such as a polypeptide having acyl-ACP reductase (AAR) activity, encoded by, for example, an aar gene.
• acyl-ACP reductase polypeptides useful in accordance with this embodiment include, but are not limited to, acyl-ACP reductase from Synechococcus elongatus PCC 7942 (GenBank YP_400611) and related polypeptides described in PCT Publication No.
• the branched fatty aldehyde is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide, such as a polypeptide having acyl-CoA reductase activity (e.g., EC 1.2.1.x), encoded by, for example, an acrl gene.
• acyl-CoA reductase polypeptides useful in accordance with this embodiment include, but are not limited to, ACR1 from Acinetobacter sp. strain ADP1 (GenBank YP_047869) and related polypeptides described in PCT Publication No. WO 2010/042664 which is incorporated by reference herein.
• the recombinant microbial cell further comprises
• polynucleotides encoding a thioesterase and an acyl-CoA synthase.
• the recombinant microbial cell produces a branched fatty alcohol (e.g., an anteiso-branched fatty alcohol or an iso-branched fatty alcohol ).
• a branched fatty aldehyde produced by the recombinant microbial cell is converted to the branched fatty alcohol.
• the recombinant microbial cell comprises a polynucleotide encoding a polypeptide (i.e., an enzyme) having fatty alcohol biosynthesis activity (also referred to herein as a "fatty alcohol biosynthesis polypeptide” or a "fatty alcohol biosynthesis enzyme”), and the branched fatty alcohol is produced by a reaction catalyzed by the fatty alcohol biosynthesis enzyme expressed or overexpressed in the recombinant microbial cell.
• a polypeptide i.e., an enzyme
• fatty alcohol biosynthesis activity also referred to herein as a "fatty alcohol biosynthesis polypeptide” or a "fatty alcohol biosynthesis enzyme”
• a composition comprising branched fatty alcohols (also referred to herein as a "branched fatty alcohol composition”), produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the polynucleotides, is recovered from the cell culture.
• the recombinant cell produces a branched fatty alcohol composition comprising anteiso- branched fatty alcohols.
• the branched fatty alcohol is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a polypeptide having fatty alcohol biosynthesis activity such as alcohol dehydrogenase (aldehyde reductase) activity, e.g., EC 1.1.1.1.
• alcohol dehydrogenase polypeptides useful in accordance with this embodiment include, but are not limited to, E. coli alcohol dehydrogenase YqhD (GenBank AP_003562) and related polypeptides described in PCT Publication Nos. WO 2007/136762 and WO2008/119082 which are incorporated by reference herein.
• the recombinant microbial cell further comprises a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase.
• the branched fatty alcohol is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a fatty alcohol biosynthesis polypeptide, such as a polypeptide having fatty alcohol forming acyl-CoA reductase (FAR) activity, e.g., EC 1.1.1.x.
• FAR polypeptides useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication No. WO 2010/062480 which is incorporated by reference herein.
• the recombinant microbial cell further comprises polynucleotides encoding a thioesterase and an acyl-CoA synthase.
• the recombinant microbial cell produces a branched
• hydrocarbon e.g., an anteiso-branched hydrocarbon or an iso-branched hydrocarbon
• a branched alkane or a branched alkene e.g., a branched terminal olefin or a branched internal olefin
• a branched fatty aldehyde produced by the recombinant microbial cell is converted into the branched hydrocarbon.
• the recombinant microbial cell comprises a polynucleotide encoding a polypeptide (i.e., an enzyme) having hydrocarbon biosynthesis activity (also referred to herein as a "hydrocarbon biosynthesis polypeptide” or a “hydrocarbon biosynthesis enzyme”), and the branched hydrocarbon is produced by a reaction catalyzed by the hydrocarbon biosynthesis enzyme expressed or overexpressed in the recombinant microbial cell.
• a polypeptide i.e., an enzyme
• hydrocarbon biosynthesis polypeptide also referred to herein as a "hydrocarbon biosynthesis polypeptide” or a "hydrocarbon biosynthesis enzyme”
• a composition comprising branched hydrocarbons (also referred to herein as a "branched hydrocarbon composition"), produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the polynucleotides, is recovered from the cell culture.
• the recombinant cell produces a branched hydrocarbon composition comprising anteiso- branched fatty hydrocarbons.
• the branched hydrocarbon is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a polypeptide having hydrocarbon biosynthesis activity such as an aldehyde decarbonylase (ADC) activity (e.g., EC 4.1.99.5), for example, a polynucleotide encoding an aldehyde decarbonylase from Prochlorococcus marinus MIT9313 (GenBank NP_895059). Additional examples of aldehyde decarbonylase and related polypeptides useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication Nos.
• ADC aldehyde decarbonylase
• Additional examples of aldehyde decarbonylase and related polypeptides useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication Nos.
• the recombinant microbial cell further comprises a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments the recombinant microbial cell further comprises a polynucleotide encoding an acyl-ACP reductase .
• a branched terminal olefin is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a hydrocarbon biosynthesis polypeptide, such as a polypeptide having decarboxylase activity as described, for example, in PCT Publication No. 2009/085278 which is incorporated by reference herein.
• the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase.
• a branched internal olefin is produced by expressing or overexpressing in the recombinant microbial cell a polynucleotide encoding a
• hydrocarbon biosynthesis polypeptide such as a polypeptide having OleCD or OleBCD activity as described, for example, in PCT Publication No. WO 2008/147781 which is incorporated by reference herein.
• the recombinant microbial cell further comprises a polynucleotide encoding a thioesterase and an acyl-CoA synthase.
• the degree of saturation of branched acyl-ACPs (which can then be converted into various branched fatty acid derivatives as described hereinabove) can be controlled by regulating the degree of saturation of fatty acid intermediates.
• the sfa, gns, and fab families of genes can be expressed, overexpressed, or expressed at reduced levels (e.g., attenuated), to control the amount of saturation of a branched acyl-ACP.
• the disclosure identifies polynucleotides useful in the recombinant microbial cells, methods, and compositions of the invention; however it will be recognized that absolute sequence identity to such polynucleotides is not necessary.
• changes in a particular polynucleotide sequence can be made and the encoded polypeptide screened for activity. Such changes typically comprise conservative mutations and silent mutations (such as, for example, codon optimization).
• Modified or mutated (i.e., mutant) polynucleotides and encoded variant polypeptides can be screened for a desired function, such as, an improved function compared to the parent polypeptide, including but not limited to increased catalytic activity, increased stability, or decreased inhibition (e.g., decreased feedback inhibition), using methods known in the art.
• the disclosure identifies enzymatic activities involved in various steps (i.e., reactions) of the BCFA biosynthetic pathways described herein according to Enzyme Classification (EC) number, and provides exemplary polypeptides (i.e., enzymes) categorized by such EC numbers, and exemplary polynucleotides encoding such polypeptides.
• exemplary polypeptides and polynucleotides which are identified herein by Accession Numbers and/or Sequence Identifier Numbers (SEQ. ID NOs), are useful for engineering BCFA pathways in parental microbial cells to obtain the recombinant microbial cells described herein.
• polypeptides and polynucleotides described herein are exemplary and non-limiting.
• sequences of homologues of representative polypeptides described herein are available to those of skill in the art using databases such as, for example, the Entrez databases provided by the National Center for Biotechnology
• NCBI National Cancer Institute
• microbial cells can be modified to contain a BCFA pathway described herein, resulting in recombinant microbial cells suitable for the production of branched chain fatty acid derivatives. It is also understood that a variety of cells can provide sources of genetic material, including sequences of polynucleotides encoding polypeptides suitable for use in a recombinant microbial cell provided herein.
• polypeptides i.e., enzymes having activities suitable for use in the BCFA biosynthetic pathways described herein.
• polypeptides are collectively referred to herein as "BCFA pathway polypeptides"
• BCFA pathway enzymes BCFA pathway enzymes
• BCFA pathway polypeptides suitable for use in recombinant microbial cells of the invention are provided in the Tables and Description and in the Examples herein.
• the invention includes a recombinant microbial cell comprising a polynucleotide sequence (also referred to herein as a "BCFA pathway polynucleotide” sequence) which encodes a BCFA pathway polypeptide.
• a polynucleotide sequence also referred to herein as a "BCFA pathway polynucleotide” sequence
• BCFA pathway polypeptides and polynucleotides encoding them suitable for use in engineering a BCFA pathway in a recombinant microbial cell of the invention can be obtained by a number of methods.
• EC numbers classify enzymes according to the reaction catalyzed.
• Enzymes that catalyze a reaction in a biosynthetic pathway described herein can be identified by searching the EC number corresponding to that reaction in a database such as, for example: the KEGG database (Kyoto Encyclopedia of Genes and Genomes; Kyoto University and University of Tokyo); the UNIPROTKB database or the ENZYME database (ExPASy Proteomics Server; Swiss Institute of Bioinformatics); the PROTEIN database or the GENE database (Entrez databases; National Center for Biotechnology Information (NCBI)); or the BRENDA database (The Comprehensive Enzyme Information System; Technical University of Braunschweig); all of which are available on the World Wide Web.
• a database such as, for example: the KEGG database (Kyoto Encyclopedia of Genes and Genomes; Kyoto University and University of Tokyo); the UNIPROTKB database or the ENZYME database (ExPASy Proteomics Server; Swiss Institute of Bioinformatic
• a BCFA pathway polynucleotide encoding a BCFA pathway polypeptide having an enzymatic activity categorized by an EC number (such as, an EC number listed in the Description or in one of Tables herein), or a fragment or a variant thereof having that activity is used in engineering the corresponding step of a BCFA pathway in a recombinant microbial cell.
• a BCFA pathway polynucleotide sequence encodes a polypeptide which is endogenous to the parental cell of the recombinant cell being engineered. Some such endogenous polypeptides are overexpressed in the recombinant microbial cell.
• An "endogenous polypeptide", as used herein, refers to a polypeptide which is encoded by the genome of the parental (e.g, wild-type) cell that is being engineered to produce the recombinant microbial cell.
• a BCFA pathway polypeptide such as for example an endogenous BCFA pathway polypeptide
• overexpress means to express or cause to be expressed a polynucleotide or polypeptide in a cell at a greater concentration than is normally expressed in a corresponding parental (for example, wild-type) cell under the same conditions.
• a polypeptide is "overexpressed" in a recombinant microbial cell when it is present in a greater concentration in the
• recombinant cell as compared to its concentration in a non-recombinant host cell of the same species (e.g., the parental cell) when cultured under the same conditions.
• the BCFA pathway polynucleotide sequence encodes an exogenous or heterologous polypeptide.
• the polypeptide encoded by the polynucleotide is exogenous to the parental microbial cell.
• An "exogenous" (or
• heterologous polypeptide refers to a polypeptide not encoded by the genome of the parental (e.g, wild-type) microbial cell that is being engineered to produce the recombinant microbial cell. Such a polypeptide can also be referred to as a "non-native" polypeptide. A variant (that is, a mutant) polypeptide is an example of an exogenous polypeptide.
• a BCFA pathway polypeptide comprises an amino acid sequence other than that of one of the exemplary polypeptides provided herein; for example, the BCFA pathway polypeptide can comprise a sequence which is a homologue, a fragment, or a variant of the sequence of the exemplary polypeptide.
• homolog refers to a polynucleotide or a polypeptide comprising a sequence that is at least 50%, preferably at least 60%, more preferably at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous to the corresponding polynucleotide or polypeptide sequence.
• a polynucleotide or a polypeptide comprising a sequence that is at least 50%, preferably at least 60%, more preferably at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous to the corresponding polyn
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or polynucleotide sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of a second sequence.
• amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared.
• a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity” is equivalent to amino acid or nucleic acid "homology”).
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the comparison of sequences and determination of percent homology (i.e., percent identity) between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)).
• the percent homology between two amino acid sequences also can be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)).
• the percent homology between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a
• a preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• an “equivalent position” (for example, an “equivalent amino acid position” or “equivalent nucleic acid position”) is defined herein as a position (such as, an amino acid position or nucleic acid position) of a test polypeptide (or test polynucleotide) sequence which aligns with a corresponding position of a reference polypeptide (or reference polynucleotide) sequence, when optimally aligned using an alignment algorithm as described herein.
• the equivalent amino acid position of the test polypeptide need not have the same numerical position number as the corresponding position of the reference polypeptide; likewise, the equivalent nucleic acid position of the test polynucleotide need not have the same numerical position number as the corresponding position of the reference polynucleotide.
• the BCFA pathway polypeptide is a variant of a reference (e.g., a parent) polypeptide, such as a variant of an exemplary BCFA pathway polypeptide described herein.
• a "variant" (alternatively, “mutant”) polypeptide as used herein refers to a polypeptide having an amino acid sequence that differs from that of a parent (e.g., wild- type) polypeptide by at least one amino acid.
• the variant can comprise one or more conservative amino acid substitutions, and/or can comprise one or more non-conservative substitutions, compared to the parent polypeptide sequence.
• the variant polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid substitutions, additions, insertions, or deletions compared to the parent polypeptide sequence.
• the sequence of the variant polypeptide is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the parent polypeptide.
• the BCFA pathway polypeptide is a fragment of a reference (e.g., a parent) polypeptide, such as a fragment of an exemplary BCFA pathway polypeptide described herein.
• fragment refers to a shorter portion of a full-length polypeptide or protein ranging in size from four amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the invention, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).
• a homologue, a variant, or a fragment further comprises one or more sequence motifs as defined herein.
• polypeptide further comprises one or more sequence motifs selected from SEQ ID NOs: 15- 21.
• a homologue, a variant, or a fragment of a branched chain alpha-keto acid dehydrogenase El-beta subunit polypeptide further comprises one or more sequence motifs selected from SEQ ID NOs: 36-42.
• a homologue, a variant, or a fragment of a lipoamide acyltransferase polypeptide further comprises one or more sequence motifs selected from SEQ ID NOs: 57-62.
• a homologue, a variant, or a fragment of a dihydrolipoyl dehydrogenase polypeptide further comprises one or more sequence motifs selected from SEQ ID NOs: 79-83.
• a homologue, a variant, or a fragment of a beta-ketoacyl-ACP synthase III polypeptide further comprises one or more sequence motifs selected from SEQ ID NOs:110- 115. Determination that a sequence contains a particular sequence motif can be readily accomplished, for example, using the ScanProsite tool available on the World Wide Web site of the ExPASy Proteomics Server.
• a BCFA polypeptide may have conservative or non-essential amino acid substitutions, relative to a parent polypeptide, which does not have a substantial effect on a biological function or property of the BCFA polypeptide. Whether or not a particular substitution will be tolerated (i.e., will not adversely affect a desired biological function, such as enzymatic activity) can be determined, for example, as described in Bowie et al. (Science, 247: 1306-1310 (1990)).
• a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• variants can be naturally occurring or created in vitro.
• variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion procedures, or standard cloning techniques.
• variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.
• variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications (including, but not limited to, increased catalytic activity (turnover number), improved stability, and reduced feedback inhibition). In such procedures, a large number of modified nucleic acid sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates. For example, variants can be prepared by using random or site-directed
• Variants can also be created by in vivo mutagenesis.
• random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways.
• a bacterial strain such as an E. coli strain
• Such "mutator" strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence in one of these strains will eventually generate random mutations within the DNA.
• Mutator strains suitable for use for in vivo mutagenesis are described in, for example, International Patent Application Publication No. WO 1991/016427.
• Variants can also be generated using cassette mutagenesis.
• cassette mutagenesis a small region of a double-stranded DNA molecule is replaced with a synthetic
• oligonucleotide "cassette" that differs from the native sequence.
• the oligonucleotide often contains a completely and/or partially randomized native sequence.
• Recursive ensemble mutagenesis can also be used to generate variants.
• Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypicaily related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis.
• Recursive ensemble mutagenesis is described in, for example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89: 7811- 7815 (1992).
• variants are created using exponential ensemble
• Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993).
• Preferred fragments or variants of a parent polypeptide retain some or all of a biological function or property (such as, enzymatic activity, thermal stability) of the parent polypeptide.
• the fragment or variant retains at least 75% (e.g., at least 80%, at least 90%, or at least 95%) of a biological function or property of the parent polypeptide.
• the fragment or variant retains about 100% of a biological function or property of the parent polypeptide.
• the fragment or variant of the parent polypeptide exhibits an increased catalytic activity (as reflected by, for example, a higher turnover number, an altered pH optimum, or a decreased K m for a desired substrate), relative to that of the parent polypeptide, under conditions in which the recombinant microbial cell is cultured.
• the parent polypeptide is endogenous to (that is, is derived from) a thermophilic cell, and if the recombinant microbial cell is generally cultured at a lower temperature than the thermophilic cell, the parent polypeptide may exhibit significantly reduced activity at the lower temperature; in which case, the variant polypeptide preferably exhibits an increased catalytic activity (such as, a higher turnover number), relative to that of the parent polypeptide, at that lower temperature.
• the fragment or variant of the parent polypeptide exhibits improved stability, relative to that of the parent polypeptide, under conditions in which the recombinant microbial cell is cultured.
• stability can include stability towards changes in temperature, ionic strength, pH, or any other differences in growth or media conditions between the recombinant microbial cell and the cell from which the parent polypeptide was derived.
• the parent polypeptide is derived from a psychrotrophic cell, and if the recombinant microbial cell is generally cultured at a higher temperature than the psychrotrophic cell, the parent polypeptide may be relatively unstable at the higher temperature ; in which case, the variant polypeptide preferably exhibits improved stability relative to that of the parent polypeptide at that higher temperature.
• the fragment or variant of the parent polypeptide exhibits reduced inhibition of catalytic activity (such as, reduced feedback inhibition) by a cellular metabolite or by a culture media component, relative to such inhibition exhibited by the parent polypeptide, under conditions in which the recombinant microbial cell is cultured.
• a BCFA pathway polypeptide is a homologue, a fragment, or a variant of a parent polypeptide, wherein the BCFA pathway polypeptide is effective in carrying out a BCFA pathway reaction in a recombinant microbial cell.
• a BCFA pathway polypeptide is suitable for use in a recombinant microbial cell of the invention.
• test polypeptide such as, for example, a BCFA pathway polypeptide described herein, or a homologue, a fragment, or a variant thereof
• a test polypeptide such as, for example, a BCFA pathway polypeptide described herein, or a homologue, a fragment, or a variant thereof
• a host cell is engineered to obtain a parental cell that comprises all the activities necessary to catalyze the reactions of the biochemical pathway in question, except for the specific pathway reaction being tested (although, in some instances, the parental cell may express endogenous polypeptide(s) that catalyze the specific pathway reaction being tested; in such instances the endogenous activity will preferably be low enough to readily detect an increase in product owing to the activity of the test polypeptide).
• a polynucleotide encoding the test polypeptide, operatively linked to a suitable promoter is then introduced into the parental cell, generating a test cell.
• the test cell and the parental cell are cultured separately under identical conditions which are sufficient for expression of the pathway polypeptides in the parental and test cell cultures and expression of the test polypeptide in the test cell culture.
• samples are obtained from the test cell culture and the parental cell culture.
• the samples are analyzed for the presence of a particular pathway intermediate or product. Presence of the pathway intermediate or product can be determined by methods including, but not limited to, gas chromatography (GC), mass spectroscopy (MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography (LC), GC coupled with a flame ionization detector (FID), GC-MS, and LC-MS.
• GC gas chromatography
• MS mass spectroscopy
• TLC thin layer chromatography
• HPLC high-performance liquid chromatography
• LC liquid chromatography
• FID flame ionization detector
• GC-MS flame ionization detector
• Example 11 herein provides methods of analyzing culture samples for the presence of a BCFA pathway intermediate or product, such as a branched fatty acid, a branched fatty alcohol, a branched fatty ester or a branched hydrocarbon.
• a BCFA pathway intermediate or product such as a branched fatty acid, a branched fatty alcohol, a branched fatty ester or a branched hydrocarbon.
• test polypeptide is effective in carrying out a BCFA pathway reaction and is suitable for use in a recombinant microbial cell of the invention.
• the invention includes a method of making a branched fatty acid derivative composition, the method comprising culturing a recombinant microbial cell of the invention in a culture medium containing a carbon source under conditions effective to express the recombinant polynucleotide sequences, and optionally isolating the produced branched fatty acid derivative composition.
• a "branched fatty acid derivative composition” is a composition comprising a branched fatty acid derivative as defined herein, such as, for example, a branched fatty acid, a branched fatty ester (e.g., a branched fatty methyl ester, a branched fatty ethyl ester , a branched wax ester), a branched fatty aldehyde, a branched fatty alcohol, a branched hydrocarbon (such as a branched alkane, a branched alkene, a branched terminal olefin, a branched internal olefin), or a branched ketone.
• a "branched fatty acid derivative as defined herein such as, for example, a branched fatty acid, a branched fatty ester (e.g., a branched fatty methyl ester, a branched fatty ethyl ester ,
• composition is a composition comprising a branched fatty acid, and so on.
• the invention includes a method of making a composition comprising a branched fatty acid derivative, the method comprising: obtaining a recombinant microbial cell (such as, a culture comprising a recombinant microbial cell) comprising : (a)
• polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched-chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydrolipoamide dehydrogenase activity, and (b) a polynucleotide encoding a polypeptide having beta-ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate, wherein at least one polynucleotide according to (a) or (b) encodes a polypeptide that is exogenous to the parental microbial cell or expression of said polynucleotide is modulated in the recombinant microbial cell; the recombinant microbial cell further comprising one or more polynucleotides each which encodes a polypeptide having fatty acid derivative enzyme activity, wherein
• recombinant microbial cell produces a branched chain fatty acid derivative when cultured in the presence of a carbon source under conditions effective to express the polynucleotides; culturing the recombinant microbial cell in a culture medium containing a carbon source under conditions effective to express the polynucleotides and produce a fatty acid derivative composition comprising straight-chain fatty acid derivatives and branched fatty acid derivatives, the branched fatty acid derivatives comprising iso-branched fatty acid derivatives and/or anteiso-branched fatty acid derivatives; and optionally recovering the composition from the culture medium.
• the fatty acid derivative composition produced by the recombinant cell comprises branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% by weight of the fatty acid derivatives in the composition are branched fatty acid derivatives.
• the fatty acid derivative composition comprises branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 15 mg/L, at least 20 mg/L, at least 25 mg/L, at least 50 mg/L, at least 75 mg/L, at least 100 mg/L, at least 125 mg/L, at least 150 mg/L, at least 175 mg/L, at least 200 mg/L, at least 225 mg/L, at least 250 mg/L, at least 275 mg/L, at least 300 mg/L, at least 325 mg/L, at least 350 mg/L, at least 375 mg/L, at least 400 mg/L, at least 425 mg/L, at least 450 mg/L, at least 475 mg/L, at least 500 mg/L, at least 525 mg/L, at least 550 mg/L, at least 575 mg/L, at least 600 mg/L, at least 625 mg/L, at least 650 mg/L, at least 10 mg/
• the fatty acid derivative enzyme activity comprises a thioesterase activity, an ester synthase activity, a fatty aldehyde biosynthesis activity, a fatty alcohol biosynthesis activity, a ketone biosynthesis activity, and/or a hydrocarbon biosynthesis activity.
• the recombinant microbial cell comprises polynucleotides encoding two or more polypeptides, each polypeptide having a fatty acid derivative enzyme activity.
• the one or more polypeptides having fatty acid derivative enzyme activity as described hereinabove wherein the recombinant microbial cell produces a composition comprising branched fatty acids, branched fatty esters, branched wax esters, branched fatty aldehydes, branched fatty alcohols, branched alkanes, branched alkenes, branched internal olefins, branched terminal olefins, or branched ketones.
• the invention includes a method of making a composition comprising an anteiso-branched fatty acid derivative, the method comprising: obtaining a recombinant microbial cell (such as, a culture comprising a recombinant microbial cell) comprising : (a) polynucleotides encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, comprising polypeptides having branched-chain alpha-keto acid
• a recombinant microbial cell such as, a culture comprising a recombinant microbial cell
• BKD branched chain alpha-keto acid dehydrogenase
• dehydrogenase activity and (b) a polynucleotide encoding a polypeptide having beta- ketoacyl-ACP synthase activity that utilizes a branched acyl-CoA molecule as a substrate; and further comprising (c) polynucleotides encoding polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deaminase activity, or (d) polynucleotides encoding polypeptides having (R)-citramalate synthase activity , isopropylmalate isomerase activity, and beta- isopropyl malate dehydrogenase activity, or (c) and (d); and (e) polypeptides having acetohydroxyacid synthase activity, acetohydroxyacid isomeroreductase activity, and di
• recombinant microbial cell produces an anteiso-branched chain fatty acid derivative when cultured in the presence of a carbon source under conditions effective to express the polynucleotides; culturing the recombinant microbial cell in a culture medium containing a carbon source under conditions effective to express the polynucleotides and produce a fatty acid derivative composition comprising straight-chain fatty acid derivatives and branched fatty acid derivatives, the branched fatty acid derivatives comprising anteiso-branched fatty acid derivatives; and optionally recovering the composition from the culture medium.
• the fatty acid derivative composition produced by the recombinant microbial cell culture comprises anteiso-branched fatty acid derivatives, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% by weight of the branched fatty acid derivatives in the
• the fatty acid derivative composition comprises anteiso-branched fatty acid derivatives in an amount (e.g., a titer) of at least 10 mg/L, at least 15 mg/L, at least 20 mg/L, at least 25 mg/L, at least 50 mg/L, at least 75 mg/L, at least 100 mg/L, at least 125 mg/L, at least 150 mg/L, at least 175 mg/L, at least 200 mg/L, at least 225 mg/L, at least 250 mg/L, at least 275 mg/L, at least 300 mg/L, at least 325 mg/L, at least 350 mg/L, at least 375 mg/L, at least 400 mg/L, at least 425 mg/L, at least 450 mg/L, at least 475 mg/L, at least 500 mg/L, at least 525 mg/L, at least 550 mg/L, at least 575 mg/L, at least 600 mg/
• an amount e.g., a titer
• the fatty acid derivative enzyme activity comprises a thioesterase activity, an ester synthase activity, a fatty aldehyde biosynthesis activity, a fatty alcohol biosynthesis activity, or a hydrocarbon biosynthesis activity.
• the recombinant microbial cell comprises polynucleotides encoding two or more polypeptides, each polypeptide having a fatty acid derivative enzyme activity.
• the recombinant microbial cell expresses or overexpresses one or more polypeptides having fatty acid derivative enzyme activity, as described hereinabove, wherein the recombinant microbial cell produces a composition comprising anteiso-branched fatty acids, anteiso-branched fatty esters, anteiso-branched wax esters, anteiso-branched fatty aldehydes, anteiso-branched fatty alcohols, anteiso- branched alkanes, anteiso-branched alkenes, anteiso-branched internal olefins, anteiso- branched terminal olefins, or anteiso-branched ketones.
• the branched fatty acid derivatives (including iso-branched fatty acid derivatives and anteiso-branched fatty acid derivatives) produced by the methods of invention may be recovered or isolated from the recombinant microbial cell culture.
• isolated refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors.
• the branched fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the branched fatty acids and derivatives thereof can collect in an organic phase either intracellular ⁇ or extracellularly.
• the collection of the products in the organic phase can lessen the impact of the branched fatty acid derivative, e.g., branched fatty aldehyde or branched fatty alcohol on cellular function and can allow the recombinant microbial cell to produce more product.
• the branched fatty acid derivative e.g., branched fatty aldehyde or branched fatty alcohol
• the branched fatty acid derivatives produced by the methods of invention are purified.
• purify means the removal or isolation of a molecule from its environment by, for example, isolation or separation.
• substantially purified molecules are at least about 60% free (e.g., at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample.
• the removal of contaminants can result in an increase in the percentage of a branched fatty acid derivative (such as, a branched fatty acid or a branched fatty aldehyde or a branched fatty alcohol or a branched fatty ester or a branched hydrocarbon) relative to other components in a sample.
• a branched fatty aldehyde or a branched fatty alcohol when a branched fatty aldehyde or a branched fatty alcohol is produced in a recombinant microbial cell , the branched fatty aldehyde or branched fatty alcohol can be purified by the removal of recombinant microbial cell proteins. After purification, the percentage of the branched fatty aldehyde or branched fatty alcohol in the sample relative to other components is increased.
• branched fatty acid or branched fatty acid derivative e.g., a branched fatty aldehyde, a branched fatty alcohol, and so forth
• a purified branched fatty acid or derivative is a branched fatty acid or derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).
• the branched fatty acid derivative may be present in the extracellular environment, or it may be isolated from the extracellular environment of the recombinant microbial cell. In certain embodiments, a branched fatty derivative thereof is secreted from the recombinant microbial cell . In other embodiments, a branched fatty acid derivative is transported into the extracellular environment. In yet other embodiments, the branched fatty acid derivative is passively transported into the extracellular environment. A branched fatty acid derivative can be isolated from a recombinant microbial cell using methods known in the art.
• Fatty acid derivatives can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14 C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Patent 7,169,588).
• biosourced carbon e.g., glucose vs. glycerol
• organic compounds or chemicals comprising both biologically- based and petroleum-based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum-based materials.
• the materials prepared in accordance with the inventive methods may be followed in commerce on the basis of their unique carbon isotope profile.
• Fatty acid derivatives produced by recombinant microbial cells can be distinguished from petroleum-based organic compounds by comparing the stable carbon isotope ratio ( C/ C) in each fuel.
• the C / C ratio in a given fatty acid derivative thereof produced according to the methods of the invention is a consequence of the 13 C/ 12 C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C 3 plants (the broadleaf), C 4 plants (the grasses), and marine carbonates all show significant differences in 13 C/ 12 C and the corresponding 6 13 C values. Furthermore, lipid matter of C 3 and C 4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway.
• the 1 C measurement scale was originally defined by a zero set by Pee Dee
• a fatty acids or derivative thereof produced according to the methods of the invention has a 6 13 C of about -30 or greater, about -28 or greater, about -27 or greater, about -20 or greater, about -18 or greater, about -15 or greater, about -13 or greater, or about -10 or greater.
• a fatty acids or derivative thereof has a 5 13 C of about -4 or less, about -5 or less, about -8 or less, about -10 or less, about -13 or less, about -15 or less, about -18 or less, or about -20 or less.
• the fatty acids or derivative thereof can have a 6 13 C bounded by any two of the above endpoints.
• a fatty acids or derivative thereof can have a 6 13 C of about -30 to about -15, about -27 to about -19, about -25 to about -21, about -15 to about -5, about -13 to about -7, or about -13 to about -10.
• a fatty acids or derivative thereof can have a 6 13 C of about -10, -11, -12, or -12.3.
• a fatty acids or derivative thereof has a ⁇ C of about -15.4 or greater.
• a fatty acids or derivative thereof has a 6 13 C of about -15.4 to about -10.9, or a 6 13 C of about -13.92 to about -13.84.
• a fatty acid derivative produced by a recombinant microbial cell can also be distinguished from petroleum-based organic compounds by comparing the amount of 14 C in each compound. Because 14 C has a nuclear half life of 5730 years, petroleum based fuels containing "older” carbon can be distinguished from fatty acids or derivatives thereof which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles", Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., Vol. I of the lUPAC Environmental Analytical Chemistry Series, Lewis Publishers, Inc., pp. 3-74 (1992)).
• f M fraction of modern carbon
• SRMs National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxl and HOxll, respectively.
• the fundamental definition relates to 0.95 times the 14 C / 12 C isotope ratio HOxl (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-lndustrial Revolution wood.
• f M is approximately 1.1.
• a fatty acid derivative produced according to the methods of the invention has a f M 14 C of at least about 1, e.g., at least about 1.003, at least about 1.01, at least about 1.04, at least about 1.111, at least about 1.18, or at least about 1.124.
• the fatty acid or derivative has an f M 14 C of about 1.130 or less, e.g., about 1.124 or less, about 1.18 or less, about 1.111 or less, or about 1.04 or less.
• the fatty acid or derivative can have a f 1 C bounded by any two of the above endpoints.
• the fatty acid or derivative can have a f M 14 C of about 1.003 to about 1.124, a f M 14 C of about 1.04 to about 1.18, or a f M 1 C of about 1.111 to about 1.124.
• M9 minimal media 6 g/L Na 2 HP0 4 , 3 g/L KH 2 P0 4 , 0.5 g/L NaCI, 1 g/L NH 4 CI, 1 mg/L thiamine, 1 mM MgS0 4 , 0.1 mM CaCl 2 .
• FA-2 media M9 supplemented with Bis-Tris buffer (0.2 M), Triton X-100 (0.1% v/v), and trace minerals containing no iron (2mg/L ZnCl. 4H 2 0, 2mg/L CaCI 2 , 6H 2 0, 2mg/L
• Che-9 media M9 supplemented with extra NH 4 CI (an additional 1 g/L), Bis-Tris buffer (0.2 M), Triton X-100 (0.1% v/v), and trace minerals (27mg/L FeCI 3 .6 H 2 0, 2mg/L ZnCI.4H 2 0, 2mg/L CaCI 2 . 6H 2 0, 2mg/L Na 2 Mo0 4 .2H 2 0, 1.9mg/L CuS0 4 .5H 2 0, 0.5mg/L H 3 B0 3 , 100 mL/L concentrated HCI).
• V9-C media Che-9 without FeCl 3 .6H 2 0
• Che-9 2N-BT media Che-9 supplemented with 20 g/L (2% w/v) glucose.
• 4NBT Che-9 supplemented with 40 g/L (4% w/v) glucose.
• This example describes the construction of a recombinant microbial cell in which the expression of a fatty acid degradation enzyme is attenuated.
• The/odE gene of E.coli also known as yafH
• yafH which encodes an acyl coenzyme A dehydrogenase (GenBank Accession No. AAC73325) involved in fatty acid degradation
• ACATATT GATTCCGGGGATCCGTCGACC (SEQ ID NO:238);
• the De ⁇ -fadE-F and De ⁇ -fadE-R primers were used to amplify the kanamycin resistance (Km R ) cassette from plasmid pKD13 (Datsenko ef al., supra) by PCR. The PCR product was then used to transform electrocompetent E.
• This example describes the construction of a recombinant microbial cell in which the expression of a fatty acid degradation enzyme and the expression of an outer membrane protein receptor are attenuated.
• the tonA (also known as fhuA) gene of E.coli MG1655 which encodes a ferrichrome outer membrane transporter which also acts as a
• bacteriophage receptor GenBank Accession No. NP_414692 was deleted from strain Dl (described above) using the Red system according to Datsenko et al., supra, with the following modifications:
• the primers used to create the tonA deletion were:
• De ⁇ -tonA-R 5'-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGAAG GTTGCGG TTGTAGGCTGGAGCTGCTTC SEQ ID NO:243
• the De ⁇ -tonA-f and Del-tonA-R primers were used to amplify the kanamycin resistance (Km R ) cassette from plasmid pKD13 by PCR.
• the PCR product obtained in this way was used to transform electrocompetent E. coli MG1655 Dl cells containing pKD46 (Datsenko et al., supra), which cells had been previously induced with arabinose for 3-4 hours.
• This example describes the construction of a recombinant microbial cell comprising a polynucleotide encoding a polypeptide having a fatty acid derivative enzyme activity.
• the tesA polynucleotide sequence encoding E.coii acyl-CoA thioesterase I (EC 3.1.1.5, 3.1.2.- ; e.g., GenBank Accession AAC73596; SEQ ID NO:202) was modified to remove the leader sequence, such that the resulting 'tesA gene product was truncated by 25 amino acids and the amino acid at the original position 26, alanine, was replaced with methionine, which then became the first amino acid of the 'TesA polypeptide sequence (SEQ ID NO:204; Cho et al., J. Biol. Chem., 270:4216-4219 (1995)).
• lacl-forward G G CTG G CTG G C AT A A AT ATCTC (SEQ ID NO:313) and lacZ-reverse:
• BKD dehydrogenase
• C part (C) of the BCFA pathway shown in Figure 1
• polynucleotide sequences encoding branched chain-specific ⁇ -ketoacyl-ACP synthases i.e., FabH polypeptides
• the strains exemplified herein also comprise polynucleotide sequences encoding fatty acid derivative enzymes, such as the modified E. coli 'tesA gene which expresses a thioesterase and generates fatty acids.
• This example demonstrates that recombinant E. coli strains engineered to express a BKD complex and a branched chain ⁇ -ketoacyl-ACP synthase produce branched chain fatty acids.
• Bacillus subtilis bkd (pKZ2 plasmid)
• B. subtilis bkd genes were amplified from B. subtilis 168 genomic DNA using the following primers:
• B.s.BKD_R 5'-GCTCTCGAGTTAGTAACAGATGTCTTC-3' (SEQ ID NO:246);
• Primers B.s.BKD_F(4g) and B.s.BKD_R amplified genes bkdAA (encoding the alpha subunit of the El component, UniProtKB P37940, GenBank NP_390285; SEQ ID NO:l ), bkdAB (encoding the beta subunit subunit of the El component, UniProtKB P37941, GenBank NP_390284 ; SEQ ID NO:22), bkdB (encoding the E2 component, UniProtKB P37942, GenBank NP_390283; SEQ ID NO:43), and IpdV (encoding the E3 component, UniProtKB P54533, GenBank NP_390286.2; SEQ ID NO:63).
• PCR products were cloned into vector pGL10.173B (SEQ ID NO:228), a pBR322 based plasmid with a P trc promoter, to produce the pKZ2 plasmid. Correct insertion of the PCR products was verified using diagnostic restriction enzyme digests.
• P. putida bkd genes were amplified from P. putida Fl genomic DNA using the following primers:
• P.p.BKDFusion_F 5'-ATAAACCATGGATCCATGAACGAGTACGCCCC-3'(SEQ ID NO:248
• P.pBKDFusion_R 5'-CCAAGCTTCGAATTCTCAGATATGCAAGGCGTG-3'(SEQ ID NO:249
• Primers P.p.BKDFusion_F and P.p.BKDFusion_R amplified P. putida genes Pput_1450 (encoding the E3 component, UniProtKB Accession No. A5W0E08; SEQ ID NO:67),
• Pput_1451 (encoding the E2 component, UniProtKB Accession No. A5W0E9; SEQ ID NO:47), Pput_1452 and Pput_1453 (encoding the El alpha and El beta subunits, UniProtKB A5W0F1 and A5W0F0, SEQ ID NOs:5 and 26, respectively).
• the PCR products were cloned into vector pGL10.173B (a pBR322 based plasmid with a Ptrc promoter; SEQ ID NO:228) to produce the pKZ4 plasmid (SEQ ID NO:231). Correct insertion of the PCR products was verified using diagnostic restriction enzyme digests.
• L. monocytogenes bkd genes were amplified from L. monocytogenes Li23 (ATCC 19114D-5) genomic DNA using the following primers:
• primer 81 (BKD_forward) GAGGAATAAACCGTGGCAACAGAATATGATGTCGTTATTCT (SEQ ID NO:250)
• primer 82 (BKD_reverse) CCCAAGCTTCGAATTTTAATACAATGCTGTATTTTCTTTGGAAAT (SEQ ID NO:251)
• the L. monocytogenes bkd operon (SEQ ID NO:232) generated by PCR was cloned into the Ncol and EcoRI sites of pGL10.173B (a pBR322 based plasmid with a Ptrc promoter; SEQ ID NO:228) to generate the plasmid pTB85.
• FabH plasmids pDG2 expression vector The pDG2 expression vector was the base plasmid for may of the constructs described below.
• the pCDFDuet-1 vector (Novagen/EMD Biosciences) carries the CloDF13 replicon, lacl gene and streptomycin/spectinomycin resistance gene (aadA).
• aadA streptomycin/spectinomycin resistance gene
• the pDG2 plasmid sequence is provided herein as SEQ ID NO: 229.
• B. subtilis fobHl pDG6
• B. subtilis fabH2 pDG7
• Streptomyces coelicolor fabH pDG8
• the pDG6 plasmid was constructed using the pDG2 plasmid.
• the fabHl coding sequence was amplified from Bacillus subtilis strain 168 using primers
• Both the fabHl insert and the pDG2 vector were digested with restriction enzymes Ndel and Xhol.
• the cut vector was treated with Antarctic phosphatase.
• the insert was ligated into the vector and transformed into transformation-competent E. coli cells. Clones were screened by DNA sequencing.
• the pDG6 plasmid sequence is provided herein as SEQ ID NO: 230, and expresses the B. subtilis FabHl polypeptide (SEQ ID NO:84) under the control of the EcfabH promoter.
• Plasmid pDG7 comprises a Bacillus subtilis fabH2 insert which expresses the ⁇ . subtilis FabH2 polypeptide (SEQ ID NO:86).
• Plasmid pDG8 comprises a Streptomyces coelicolor fabH insert which expresses the S. coelicolor FabH polypeptide (SEQ ID NO:96).
• Plasmid pKZ5 was constructed by cloning the Ncol-Avrll fragment of pDG6, containing BsFabHl under control of the EcfabH promoter, into the Ncol-Avrll cut vector pACYCDuet-1 (Novagen). Plasmid pKZ5 carries a chloramophenicaol resistance gene and a streptomycin/spectinomycin resistance gene.
• Plasmid pACYC-P Trc was constructed by PCR-amplifying the lacl q , P Trc promoter and terminator region from pTrcHis2A (Invitrogen, Carlsbad, CA) using primers
• pTrc_F TTTCGCGAGGCCGGCCCCGCCAACACCCGCTGACG
• pTrc_R AAGGACGTCTTAATTAATCAGGAGAGCGTTCACCGACAA SEQ ID NO:259
• the PCR product was then digested with Aatll and Nrul and insterted into plasmid pACYC177 (Rose, R.E., Nucleic Acids Res., 16:356 (1988)) digested with Aatll and Seal.
• the nucleotide sequence of the pACYC-P Trc vector is provided herein as SEQ ID NO: 233.
• a single point mutation was introduced in the PT I - C promoter of the pACYC-Pr rc vector to generate the variant promoter P Trc2 and the pACYC-P rrc2 vector.
• the wild-type P Tr c promoter sequence is provided herein as SEQ ID l ⁇ IO:234, and the P Trc2 variant promoter is provided herein as SEQ ID NO:235.
• the nucleotide sequence encoding E.coli acyl-CoA thioesterase I (TesA, EC 3.1.1.5, 3.1.2.- ; e.g., GenBank Accession AAC73596; SEQ ID NO:202) was modified to remove the leader sequence, such that the resulting 'tesA gene product was truncated by 25 amino acids and the amino acid at the original position 26, alanine, was replaced with methionine, which then became the first amino acid of the 'TesA polypeptide (SEQ ID NO:204; Cho et ai, J. Biol. Chem., 270:4216-4219 (1995)).
• DNA encoding the 'TesA polypeptide was inserted into the Ncol and EcoRI sites of the pACYC-P rrc vector and the pACYC-P r/r 2 vector, producing the pACYC-P Trc -tesA and pACYC-P TrC 2-tesA plasmids, respectively. Correct insertion of 'tesA sequence into the plasmids was confirmed by restriction digestion.
• the plasmid pDGlO was prepared using the PCR-Blunt vector (Invitrogen, Carlsbad, CA) and a C. acetobutylicum ptb_buk operon insert, wherein the ptb part represents the gene encoding C. acetobutylicum phosphotransbutyrylase (GenBank Accession AAA75486.1, SEQ ID NO:227), and the buk part represents the gene encoding C. acetobutylicum butyrate kinase (GenBank Accession JN0795, SEQ ID NO:226).
• the buk_ptb operon was amplified from C. acetobutylicum (ATCC 824) genomic DNA using primers 5'- CTTAACTTCATGTGAAAAGTTTGT-3' (SEQ ID NO:260) and 5'-
• DV2'tesA (MG1655 AfadE AtonA lachtesA) is E. coli strain DV2 which in addition expresses a leaderless 'tesA gene for production of fatty acids.
• DV2'tesA + BsfabHl is the DV2'tesA strain transformed with the pDG6 plasmid expressing the B. subtilis fabHl gene.
• DV2'tesA + BsfabHl + Bsbkd is the DV2'tesA strain transformed with the pDG6 plasmid expressing the B. subtilis fabHl gene, and the pKZ2 plasmid expressing the B. subtilis bkd operon.
• DV2'tesA + BsfabHl + Ppbkd is the DV2'tesA strain transformed with the pDG6 plasmid expressing the B. subtilis fabHl gene, and the pKZ4 plasmid expressing the P. putida bkd operon.
• Seed cultures were grown in LB supplemented with the appropriate antibiotics. After 4 hours of growth, the cultures were diluted 1:25 in Che-9 2NBT medium (2% glucose, nitrogen limited medium, 0.2 M Bis-Tris, pH 7.0, 0.1% Triton) + appropriate antibiotics and grown overnight. The cultures were then diluted in 4NBT (4% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton) to a final OD 60 o ⁇ 0.2. After 6 hours of growth, IPTG was added to a final concentration of 1 mM. At 24 hours post-induction, 1 ml of culture was extracted with 500 ⁇ ethyl acetate (containing 1% HCI), derivatized with freshly prepared TMAH and subjected to GC/MS analysis.
• Che-9 2NBT medium 2% glucose, nitrogen limited medium, 0.2 M Bis-Tris, pH 7.0, 0.1% Triton
• 4NBT 4% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0
• FFA free fatty acid
• BCFA branched chain fatty acid
• E. coli does not normally produce branched-chain fatty acids.
• Figure 4(b) is a GC/MS analysis of free fatty acids (FFA) produced by the control E. coli strain (DV2'tesA) which expresses a thioesterase gene but lacks enzymes of parts (C) and (D) of the BCFA pathway, and which shows no detectable production of branched chain fatty acids.
• FFA free fatty acids
• DVD2'tesA control E. coli strain
• the E. coli DV2'tesA strain produced about 2000 mg/L free fatty acids, with no detectable branched chain fatty acids, while the DV2'tesA +
• BsfabHl strain expressing the BsfabHl gene likewise produced about 2000 mg/L FFA, approximately 3 mg/L ( ⁇ 2%) of which was branched-chain fatty acids, essentially all of which were in the iso-branched configuration (Table 7).
• Branched fatty acid production increased dramatically when the E. coli strain was engineered to express bkd genes encoding polypeptides having branched-chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydroiipoamide dehydrogenase activity (corresponding to part (C) of the BCFA pathway), along with the exogenous fabH gene.
• bkd genes encoding polypeptides having branched-chain alpha-keto acid dehydrogenase activity, lipoamide acyltransferase activity, and dihydroiipoamide dehydrogenase activity (corresponding to part (C) of the BCFA pathway), along with the exogenous fabH gene.
• B. subtilis bkd genes expression of the B. subtilis bkd genes together with the B.
• subtilis fabHl gene produced a variety of branched fatty acid structures, including branched fatty acids with chain lengths from C13 to C17 in iso- branched (denoted " ⁇ -”) and anteiso-branched (denoted "a-”) forms.
• the following example describes the construction of recombinant E. coli strains which express exogenous genes and/or overexpress endogenous genes encoding enzymes which serve to increase metabolic flux through the intermediates a-ketobutyrate, the anteiso-branched a-keto acid intermediate a-keto- -methylvalerate, and the anteiso- branched chain primer 2-methylbutyryl-CoA by the (A.l) part of the pathway of Figure 3A, leading to the increased production of anteiso-branched acyl-ACP, and ultimately anteiso- branched fatty acid derivatives, in these recombinant cells.
• This example also describes the effect of attenuating expression of an undesired endogenous gene on BCFA production.
• the fabH gene of E. coli encoding a beta-ketoacyl-ACP synthase III, which utilizes straight-chain acyl-CoA molecules instead of branched-chain acyl-CoA molecules, was attenuated by deletion of that gene.
• This example also describes the effect on BCFA production of chromosomally integrating an exogenous BKD operon (corresponding to part (C) of the BCFA pathway of Figures 1 and 3B).
• This example describes the construction of a recombinant E. coli strain in which one of the chromosomal genes involved in threonine biosynthesis was mutated and was placed under control of a chromosomally-integrated lambda P L promoter.
• the gene was amplified from E.coli MG1655 DNA in two parts. The first part was amplified using primers TREE026 and TREE028 while the second part was amplified using TREE029 and TREE030 (Table 8). The primers used to amplify the two components contained overlapping sequences which were then used to "stitch" the individual pieces together. The two PCR products were combined in a single PCR reaction and primers TREE026 and TREE030 to amplify the entire thrA gene. Primers TREE028 and TREE029 were designed to create a mutation in the native thrA at codon 345, which resulted in an S345F variant of
• aspartokinase I SEQ. ID NO: 118. Previous work has shown that this mutation eliminates feedback inhibition by threonine in the host strain (Ogawa-Miyata,Y., et al., Biosci.
• the P L promoter was amplified using primers Km_trc_overF and TREE027 (Table 8) using plasmid pDS80 (Example 2) as a template. This fragment was then stitched to a kanamycin resistance cassette flanked by FRT sites, which was amplified from plasmid pKD13 using primers TREE025 and Km_trc_overR (Table 8). The resulting PCR product containing the KmFRT cassette and P L promoter was stitched to the thrA* PCR product. Primers TREE025 and TREE030 were used to amplify the entire KmFRT-P L -i/ir ⁇ * mutagenic cassette.
• primers also contain approximately 50 bp of homology to the integration site at the 5' end and the entire thrA gene as homology on the 3' end, targeting the cassette to the native thrA site in E. coli, which is part of an operon comprising the thrA, thrB and thrC genes.
• This mutagenic cassette was electroporated into the parental strain, E.coli DV2 (Example 1) containing the helper plasmid pKD46 expressing Red recombinase (Datsenko et al., supra). Clones containing the chromosomal integration were selected in the presence of kanamycin, and verified by diagnostic PCR.
• the kanamycin marker was then removed by expression of the pCP20 plasmid (Datsenko et al., supra). Proper integration and marker removal were verified by PCR and sequencing.
• the resulting strain in which the mutant thrA* gene and the the endogenous thrB and thrC genes were overexpressed by the chromosomally-integrated lambda P L promoter, was designated DV2 P L thrA*BC.
• tdcB E.coli catabolic threonine deaminase
• Catabolic threonine deaminase catalyzes the degradation of threonine to a-keto- butyrate (2-oxobutanoate), the first reaction of the threonine degradation/ isoleucine production pathway.
• the reaction catalyzed probably involves initial elimination of water (hence the enzyme's earlier identification as a threonine dehydratase), followed by isomerization and hydrolysis of the product with C-N bond breakage. Increased expression of this gene has been shown to dramatically increase levels of isoleucine in heterologous organisms (Guillouet S. et ai, Appl. Environ. Microbiol. 65:3100-3107 (1999)).
• threonine deaminase is relatively resistant to isoleucine feedback mechanisms (Guillouet et a I., supra).
• E.coli MG1655 genomic DNA was amplified using primers TREE020 and TREE021 (Table 8) to obtain the native tdcB gene.
• primers Chlor 1 and Chlor 4 were used to amplify an FRT-Kanamycin resistance cassette to be used for integration selection/screening as previously described.
• primers EG238 and TREE018 were used to amplify a region of homology 3' to the lacZ integration site, while primers TREE022 and TREE023 (Table 8) were used to amplify a region of homology 5' to the lacZ site.
• the plasmid pDS80 (Example 2) was used as a template to amplify a fragment containing the P L promoter by using primers TREE017 and TREE018 (Table 8). Each of these fragments were designed with overlaps for corresponding adjacent piece and were stitched together using SOEing PCR techniques.
• the resulting P L tdcB mutagenic cassette (approx. 4.3kb) contained approximately 700bp of homology to the integration site at the 5' end and 750bp of homology to the integration site at the 3' end.
• the P L tdcB mutagenic cassette was electroporated into the host strain, E.coli DV2 P L thrA*BC containing the helper plasmid, pKD46 (Datsenko et ai, supra). Clones containing the chromosomal integration were selected for in the presence of kanamycin, and verified by PC and sequencing analysis. The kanamycin marker was then removed using the pCP22 plasmid (Datsenko et al., supra). The resulting strain was designated DV2 P L thrA*BC P L tdcB.
• This example describes the construction of a recombinant microbial cell in which the B. subtilisfabHl gene was integrated into the chromosome and placed under transcriptional control of the strong constitutive T5 promoter.
• a PCR product was generated for the chromosomal integration of a loxPcat integration cassette comprising a chloramphenicol resistance gene, a T5 promoter (P T5 ), and BsfabHl coding sequence, at the site of the fadE deletion scar of DV2 P L thrA*BC.
• the individual components of the integration cassette were first PCR-amplified.
• the loxP-cat- loxP P T5 component was amplified from plasmid pl00.38 (SEQ ID NO:237) using primers TREE133 and TREE135 (Table 9).
• the BsfabHl gene was amplified from a plasmid carrying the BsfabHl gene using primers TREE134 and TREE136.
• Primers TREE133 and TREE136 contain the 5' and 3' 50 bp of homology sequence for integration.
• the primers used to amplify the components contain overlapping sequence which were then used to "stitch" the individual pieces together.
• the loxP-cat-P T s and BsfabHl PCR products were stitched together by combining both pieces in a single PCR reaction and using primers TREE133 and TREE136 to amplify the final loxPcat-P T s-BsfabHl integration cassette.
• the loxP-cat-P T5 -Ss/obf/J[ cassette was integrated using the Red recombinase system (Datsenko, et al., supra).
• the loxP-cat-P T 5-6s/obHi PCR product was used to transform electrocompetent DV2 P L -thrA *BC cells containing plasmid pKD46, which had been previously induced with arabinose for 3 - 4 hours at 30°C. Following a 3 hour 37°C outgrowth in SOC medium, cells were plated on Luria agar plates containing 17 g/mL chloramphenicol and incubated overnight at 37°C.
• Chloramphenicol-resistent colonies were screened by PCR for proper integration of loxP-cat-P T 5-6s/ofaHl.
• Primers fadE-L2 and fadE- R2 which flank the chromosomal integration site, were used to confirm the integration.
• the chloramphenicol marker gene was removed by expressing a Cre recombinase which promotes recombination between the two loxP sites that flank the chloramphenicol resistance gene.
• the plasmid pJW168 which harbors the cre recombinase gene, was transformed into strain DV2 Pi-thrA*BC loxP-cat-P T 5- BsfabHl and the marker was removed according to the method described by Palmeros ei al. (Gene 247:255-264 (2000)). The resulting strain DV2 P L -thrA *BC P J5 -BsfabHl was verified by sequencing.
• This example describes the construction of a recombinant E. coli cell in which the expression of an undesired endogenous gene (in this instance, the fabH gene of E. coli, encoding a beta-ketoacyl-ACP synthase III which utilizes straight-chain acyl-CoA molecules instead of branched-chain acyl-CoA molecules) was attenuated by deletion of that gene.
• an undesired endogenous gene in this instance, the fabH gene of E. coli, encoding a beta-ketoacyl-ACP synthase III which utilizes straight-chain acyl-CoA molecules instead of branched-chain acyl-CoA molecules
• the fabH gene of E.coli was deleted from DV2 P L -thrA *BC P JS -BsfabHl using the Red recombinase system (Datsenko ei al., supra).
• Primers TREE137 and TREE138 (Table 9), were used to amplify the kanamycin resistance cassette from plasmid pKD13 by PCR. The PCR product was then used to transform electrocompetent DV2 P_-thrA*BC P J5 -BsfabHl cells containing plasmid pKD46. Deletion of EcfabH and removal of the kanamycin marker were carried out according to the method described by Wanner and Datsenko, supra. Primers TREE139 and TREE140 were used to confirm the deletion of EcfabH. The final markerless strain was named DV2 P L -thrA *BC P J5 -BsfabHl McfabH.
• a recombinant E. coli strain was constructed containing chromosomally-integrated genes overexpressing enzymes of parts (A.l) and (D) of the anteiso-BCFA biosynthetic pathway of Figures 3A and 3B.
• the Pi-tdcB mutagenic cassette (prepared as described above) was integrated into strain DV2 P L -thrA*BC Pj S -BsfabHl McfabH to generate the strain DV2 P L -thrA *BC P L -tdcB P 75 -BsfabHl McfabH.
• coli tdcB gene are both under the control of strong lambda P L promoters, and the integrated B. subtilis fabHl gene is under the control of the strong T5 promoter.
• the endogenous E. colifadH gene was deleted from this strain.
• the pOP80 plasmid was constructed by digesting the cloning vector pCL1920 (GenBank AB236930; Lerner C.G. and Inouye M., Nucleic Acids Res. 18:4631 (1990)) with the restriction enzymes Aflll and Sfol. Three DNA fragments were produced by this digestion. The 3737 bp fragment was gel-purified using a gel-purification kit (Qiagen, Inc., Valencia, CA).
• a DNA sequence fragment containing the P Trc promoter and lacl region from the commercial plasmid pTrcHis2 was amplified by PCR using primers LF302 (5'-atatgacgtcGGCATCCGCTTACAGACA-3', SEQ ID NO:292) and LF303 (5'-aattcttaagTCAGGAGAGCGTTCACCGACAA-3', SEQ ID NO:293) introducing the recognition sites for the Zral and Aflll enzymes, respectively.
• the PCR products were purified using a PCR-purification kit (Qiagen, Inc.
• the PCR product was gel-purified and ligated with the 3737 bp DNA sequence fragment derived from pCL1920 to generate the expression plasmid pOP80 containing the P Trc promoter.
• a plasmid was constructed which expresses the ilvA gene encoding a threonine deaminase from Corynebacterium glutamicum, and was tested for its suitability for use in part (A.l) of the anteiso-BCFA biosynthetic pathway of Figure 3A.
• the genomic DNA of Corynebacterium glutamicum was used to amplify the ilvA gene using the following primers: j
• the gene was inserted into the Ncol and EcoRI sites of plasmid OP80 (above).
• the plasmid was sequence verified, then transformed into DV2 P L -thrA*BC P J5 -BsfabHl McfabH .
• This strain was tested for branched fatty acid production against DV2 P L -thrA*BC Pi-tdcB EcfabH, which has an integrated tdcB gene under control of the P L promoter.
• the strains were grown in FA-2 media following the protocol outlined below.
• FFA free fatty acid
• BCFA branched chain fatty acid
• p plasmid-expressed BsfabHl (pDG6) int - chromosomally integrated BsfabHl gene
• This example shows an increase in the amount and the proportion of BCFA produced by strain 3, in which the BsfabHl gene (encoding a polypeptide having branched chain beta-ketoacyl-ACP synthase III activity) was chromosomally integrated, compared to that produced by strain 2 containing the plasmid- expressed BsfabHl gene.
• the proportion of anteiso-BCFA produced by the strains containing chromosomally integrated and plasmid-expressed BsFabHl was relatively unchanged; about 38 to 40% of the total BCFA produced by these strains were anteiso- BCFA.
• strain 3 contained the endogenous E. coli fabH gene involved in straight-chain fatty acid production. Deletion of that gene from strain 4 significantly increased the amount of BCFA produced by that strain, increasing from about 34% of the FFA produced in branched form in strain 3, to about 45% of the FFA produced in branched form in strain 4. On the other hand, the proportion of anteiso-BCFA produced by these strains was relatively unchanged; in both of these strains, between 36% to 38% of the total BCFA was produced in the anteiso-form.
• strain 1 which did not overexpress any of the (Al) pathway activities, produced about 12% of BCFA in the anteiso-BCFA form.
• strain 2 (DV2 thrA*BC), which overexpressed polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, and threonine synthase activity, produced about 40% of BCFA in the anteiso-BCFA form. Strain 2 also shows that the native threonine deaminase activity present in the parental microbial cell was sufficient for production of anteiso- branched chain fatty acids by the anteiso-BCFA pathway shown in Fig. 3A.
• Comparing strains 5 and 6 further demonstrates the effect of manipulating beta- ketoacyl-ACP synthase III activity (step (D) of the BCFA pathway) on BCFA production.
• Both strains 5 and 6 overexpress the thrA*BC and tdcB genes.
• the proportion of BCFA produced was nearly doubled, from about 30% of the FFA by strain 5 to over 60% of the FFA by strain 6.
• strain 6 also showed an increase in the proportion of anteiso-BCFA produced (from about 57% of BCFA by strain 5, to about 66% of BCFA by strain 6), which was albeit a less dramatic relative increase than in the proportion of BCFA produced.
• Comparing strains 6 and 7 demonstrates that an enzyme that catalyzes a particular pathway reaction can be substituted by a different enzyme which catalyzes the same pathway reaction.
• an endogenous enzyme encoded by the E. coli tdcB gene that was overexpressed in strain 6 was substituted in strain 7 by an exogenous enzyme encoded by the C. glutamicum ilvA gene.
• the E. coli tdcB gene encodes a catabolic threonine deaminase
• the C. glutamicum ilvA gene encodes an anabolic threonine deaminase.
• Both of these enzymes catalyze the conversion of threonine to a-ketobutyrate (i.e., 2-oxobutanoate) and both are classified under EC 4.3.1.19.
• these enzymes are derived from different sources and are encoded by different genes, Table 10 shows that both of these enzymes are suitable for use in a recombinant microbial cell to carry out the conversion of threonine to a-ketobutyrate in the anteiso-BCFA pathway described herein: strain 6 produced about 66% of BCFA in the anteiso-form, while strain 7 produced over 70% of BCFA in the anteiso-form. This result not only confirms that the C.
• glutamicum anabolic threonine deaminase is suitable for use in a recombinant microbial cell to catalyze the conversion of threonine to a-ketobutyrate according to the anteiso-BCFA pathway, it demonstrates that a pathway enzyme (such as, a pathway enzyme described herein) which catalyzes a particular pathway reaction can be "functionally replaced" in the recombinant microbial cell by a different enzyme which catalyzes the same reaction.
• a pathway enzyme such as, a pathway enzyme described herein
• the following example describes the construction of recombinant E. coli strains which express exogenous genes and/or overexpress endogenous genes encoding enzymes which serve to increase metabolic flux through the intermediates ⁇ -ketobutyrate, the anteiso-branched a-keto acid intermediate a-keto-P-methylvalerate, and the anteiso- branched chain primer 2-methylbutyryl-CoA by the (A.2) part of the BCFA pathway of Figure 3A, leading to the increased production of anteiso-branched acyl-ACP, and ultimately anteiso-branched fatty acid derivatives, in these recombinant cells.
• This example also describes the construction of plasmids which express a fabH gene from Listeria monocytogenes and a novel mutant L monocytogenes fabH gene, which provide alternative beta-ketoacyl-ACP synthase III enzymes for part (D) of the BCFA biosynthetic pathways of Figures 1 and 3.
• DNA encoding CimA3.7 was synthesized by Geneart AG (Regensburg, Germany). The DNA was cloned into the Sfil site of plasmid pMK-RQ (kanR) (Geneart AG, Regensburg, Germany). Flanking the coding sequence, a 5' Kpnl restriction site and a 3' Sacl restriction site were introduced directly upstream of the ATG start codon and immediately
• the cimA 3.7 cloning vector was verified by sequencing.
• the individual components of the integration cassette were PCR-amplified as follows.
• the KmFRT component was amplified from plasmid pKD13 using primers TREE146 and Km_trc_overR (Table 11).
• the P Trc promoter was amplified from pOP80 (Example 4) using primers Km_trc_overF and TREE033.
• the cimA3.7 coding sequence was amplified from the cimA 3.7 cloning vector described above using primers TREE032 and TREE035.
• E. coli native leuBC genes were amplified using E. coli genomic DNA and primers TREE034 and TREE104.
• the forward primer TREE146 which was used to amplify the KmFRT cassette, included the 5' 50bp of homology sequence for integration.
• Each of the primers used to amplify the components contained overlapping sequence which were used to "stitch" the individual pieces together.
• KmFRT and P Trc were stitched together by combining both pieces in a single PCR reaction and using primers TREE146 and TREE033 to amplify the KmFRT-P Tr c product.
• KmFRT-P Tr c was then stitched with cimA3.7 using primers TREE146 and TREE035 to generate KmFRT-P Trc -c/>7?A3.7.
• the final piece, leuBC was stitched to KmFRT-P Trc -c/m/ 3.7 using primers TREE146 and TREE104 to generate the final integration cassette: Km FRT-P Trc -c/m/43.7 leuBC.
• TREE032 GTAT ATATTA ATGTATCG ATTA A ATA AG G AG G A AT A Amplify 298
• TREE034 gatttgttggctatagttagagaagttactggaaaattgTAACAAG Amplify 300
• TREE104 GG 1 AGCGAAGG 1 1 1 I GCCCGGC Amplify 302 leuBC
• the KmFRT-P Trc -c/ ' /77A3.7 /ei/6C cassette was integrated into the £ coli genome using the Red recombinase system (Datsenko et oi, supra).
• the KmfRT-P Jrc -cimA3.7 leuBC PCR product was used to transform electrocompetent £ coli MG1655 DV2 cells containing plasmid pKD46, which had been previously induced with arabinose for 3 - 4 hours at 30°C. Following a 3-hour 37°C outgrowth in SOC medium, cells were plated on Luria agar plates containing 50 ⁇ g/mL kanamycin and incubated overnight at 37°C.
• Kanamycin-resistant colonies were screened by PCR for proper integration of KmFRT-P T rc-c//77A3.7.
• Primers TREE151 and TREE106 which flank the chromosomal integration site, were used to confirm the integration.
• the kanamycin marker gene was removed in accordance with the method described by Datsenko et al., supra.
• the genomic DNA of Listeria monocytogenes Li23 was used as template to amplify the fabH gene using the following primers:
• TREE044 (fabHjorward) G AG G A ATA A ACC ATG A ACG C AG G A ATTTTAG G AG TAG (SEQ ID NO:256); primer 61 (fabH_reverse)
• the PCR product was then cloned into the Ncol/ EcoRI sites of pDS80 (a pCL1920- based vector carrying the phage lambda P L promoter; SEQ ID NO:236) and transformed into transformation-competent E. coli cells. Individual colonies were picked for sequence verification of cloned inserts.
• the nucleic acid sequence of wild type L monocytogenes fabH (SEQ ID NO:99) encodes the wild type LmFabH l protein (SEQ ID NO:98), and the plasmid containing this sequence was designated pTB.079.
• a mutant L. monocytogenes fabH gene was discovered containing a T to G change at position 928, resulting in a change in the expressed protein at amino acid position 310 from Tryptophan (W) to Glycine (G) , i.e., a W310G variant.
• the novel mutant L monocytogenes fabH gene (SEQ ID NO:101) encoding the FabH W310G variant was designated LmFabH2 (SEQ ID NO:100), and the plasmid containing this sequence pTB.081.
• Plasmids containing the wild type LmfabHl gene (pTB.079) and the mutant LmfabHl gene (pTB.081) were transformed into the DV2 P Trc -cimA3.7_leuBCD/pACYCtrc2_tes/ strain.
• the strains were transformed with pKZ4 (P. putida BKD) and pDG6 (B subtilis fabHl) plasmids and evaluated for BCFA production.
• FFA free fatty acid
• BCFA branched chain fatty acid
• Pp plasmid-expressed BKD operon from P. putida (pKZ4)
• BsHl plasmid-expressed B. subtilis FabH l (pDG6)
• LmH2 plasmid-expressed L. monocytogenes FabH2 W310G (pTB.081)
• strains 2 and 3 of Table 12 demonstrate that little if any BCFA is produced in microbial cells lacking branched chain alpha-keto acid dehydrogenase (BKD) activities and/or beta-ketoacyl-ACP synthase III (e.g., FabH) activity specific for branched-chain substrates, corresponding to steps (C) and (D) of the BCFA pathway of Figure 1.
• BKD branched chain alpha-keto acid dehydrogenase
• beta-ketoacyl-ACP synthase III e.g., FabH
• Comparing strains 1 and 4 shows the effect of engineering activities corresponding to part (A.2) of the BCFA pathway in recombinant microbial cells on the production of anteiso-BCFA .
• Strains 1 and 4 produced nearly identical amounts and proportions of BFCA (about 27% of the total FFA produced in these cells were branched fatty acids), however, strain 1 produced primarily iso-BCFA, with only about 12% of the total BCFA being in the anteiso-form.
• strain 4 expressing genes encoding polypeptides having (R)-citramalate synthase activity , isopropylmalate isomerase activity, and beta-isopropyl malate dehydrogenase activity (corresponding to part (A.2) of the BCFA pathway) produced substantially more anteiso-BCFA, with nearly 70% of the total BCFA in the anteiso-form.
• Comparing strains 4, 5 and 6 shows that a variety of polypeptides having branched chain beta-ketoacyl-ACP synthase III activity can be utilized for producing branched chain fatty acids in recombinant microbial cells. More particularly, this example shows that the L monocytogenes wild type FabH and a novel W310G variant of L. monocytogenes FabH have activities suitable for use in the BCFA pathway. Comparing FFA produced by cultures of these three strains, which are identical except for t e fabH genes employed, shows that strain 5 expressing B.
• subtilis FabHl produced about 27% of total FFA in branched form, with almost 70% of those branched fatty acids in the anteiso-form; strain 6 expressing wild- type L. monocytogenes FabH produced about 38% of total FFA in branched form, with about 75% of of those branched fatty acids in anteiso-form; and strain 7 expressing a L.
• W310G variant FabH (designated FabH2) produced about 26% of total FFA in branched form, with, remarkably, over 90% of those branched fatty acids in the anteiso- form.
• the following example describes the construction of recombinant E. coli strains which express exogenous genes and/or overexpress endogenous genes encoding enzymes which serve to increase metabolic flux through the intermediates ot-ketobutyrate, the anteiso-branched a-keto acid intermediate a-keto-P-methylvalerate, and the anteiso- branched chain primer 2-methylbutyryl-CoA by the combined (A.l) and (A.2) parts of the pathway of Figure 3A, leading to even greater production of anteiso-branched acyl-ACP, and ultimately anteiso-branched fatty acid derivatives, in these recombinant cells.
• This example also describes the construction of a plasmid which expresses bkd genes from Listeria monocytogenes, which provides another example of branched-chain alpha- keto acid dehydrogenase (BKD) complex enzymes suitable for use in part (C) of the BCFA biosynthetic pathway of Figure 1.
• BKD branched-chain alpha- keto acid dehydrogenase
• the P rc -cimA3.7_leuBCD cassette (Example 5) was integrated into strain DV2 Pi-thrA*BC P J5 -BsfabHl McfabH (Example 4) to generate the strain DV2 P L -thrA*BC P Trc - cimA3.7_leuBCD P 15 -BsfabHl EcfabH, which was also called strain Gl.
• This strain overexpressed polypeptides having (R)-citramalate synthase activity, isopropylmalate isomerase activity, and beta-isopropyl malate dehydrogenase activity according to the (A.2) part of the anteiso-BCFA pathway, and overexpressed polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, and threonine synthase activity according to the (A.l) part of the part of the anteiso-BCFA pathway.
• the P L - tdcB cassette (Example 4) was integrated into strain Gl, to generate strain DV2 Pi-thrA*BC Pi-tdcB P lfC -cimA3.7_leuBCD P J5 -BsfabHl McfabH, which was also called strain G2.
• coli tdcB gene (encoding polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and threonine deaminase activity, corresponding to the (A.l) part of the BCFA pathway) were placed under the control of strong lambda P L promoters, and were as such overexpressed.
• coli leuBCD genes (encoding polypeptides having (R)-citramalate synthase activity , isopropylmalate isomerase activity, and beta-isopropyl malate dehydrogenase activity corresponding to the (A.2) part of the BCFA pathway), were also integrated into the E. coli chromosome under control of the strong IPTG-inducible promoter P Trc and therefore were also overexpressed.
• the integrated B. subtilisfobHl gene encoding a branched chain beta ketoacyl-ACP synthase III corresponding to part (C) the pathway, was under the control of the strong T5 promoter.
• the endogenous E. colifadH gene was deleted from this strain.
• Plasmid pTB85 (expressing the L. monocytogenes BKD complex)
• the genomic DNA of Listeria monocytogenes Li23 was used for amplification of the bkd genes using the following primers:
• primer81 (BKDjor) G AG G A ATA A ACCGTG G C A AC AG A ATATG ATGTCG TTATTCT (SEQ ID NO:306)
• primer82 (BKD_rev) CCCAAGCTTCGAATTTTAATACAATGCTGTATTTTCTTTGGAAAT (SEQ ID NO:307)
• the Lmbkd PCR product was cloned into the Ncol and EcoRI sites of pGL10.173B (SEQ ID NO:228) under the control of the P trc promoter.
• the sequence-verified plasmid was transformed into strain G l (above).
• the strains were also transformed with pACYC-P TrC 2-tesA (leaderless E. coli TesA) pKZ4 (P. putida BKD) and pDG6 (B subtilis fabHl) plasmids and evaluated for BCFA production using the FA-2 media protocol as described in Example 4.
• strains DV2 P L -thrA*BC P J5 -BsfabHl, DV2 ? L -thrA *BC ⁇ -BsfabHl kEcfabH, DV2 P l -thrA*BC P t -tdcB P J5 -BsfabHl AEcfabH, Gl, and G2 were transformed with plasmids pKZ4, which expresses PpBKD, and pACYC-P T rc2-tesA which expresses the leaderless E. coli 'tesA.
• Strain DV2 Pi-thrA*BC transformed with plasmids pKZ4, pACYC-P T rc2-tes/4, and pDG6 (which expresses BsfabHl) served as a control for these experiments.
• plasmids pKZ4, pACYC-P T rc2-tes/4, and pDG6 which expresses BsfabHl
• FFA free fatty acid
• BCFA branched chain fatty acid
• Lm plasmid-expressed L monocytogenes BKD operon
• AEc deleted E. coli chromosomal fabH gene
• strain DV2 thrA*BC tdcB (strain 2 in Table 13 above), which overexpressed polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity and threonine deaminase activity (according to the (A.l) part of the anteiso-BCFA pathway) and a polypeptide having branched chain beta-ketoacyl-ACP synthase III activity by a
• chromosomally integrated BsfabHl gene produced about two-thirds (66%) of its branched chain fatty acids in the anteiso-form.
• strain Gl Comparing strain 2 to strain 3 (DV2 thrA*BC cimA3.7 leuBCD , also denoted “strain Gl”), which overexpressed polypeptides having (R)-citramalate synthase activity , isopropylmalate isomerase activity, and beta-isopropyl malate dehydrogenase activity (according to the (A.2) part of the anteiso-BCFA pathway) in addition to polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, and threonine synthase activity, the amount and proportion of BCFA produced was comparable (about 59% of FFA produced as BCFA in strain 2, compared to about 62% of FFA produced as BCFA in strain 3), but the proportion of anteiso-BCFA was much greater in strain 3 (Gl) than in strain 2, such that about 84% of the BCFA produced by strain 3 was in the anteiso-
• strain 3 Comparing strains 3 and 4 shows the effect of different BKD enzyme complexes on BCFA and anteiso-BCFA production.
• Strain 3 expressed bkd genes from P. putida while strain 4 expressed bkd genes from L monocytogenes. Although the strain expressing the L.
• monocytogenes bkd genes showed a lower overall production (titer) of both FFA and BCFA than the strain expressing the P. putida bkd genes, the proportions of anteiso-branched fatty acids produced by these strains were remarkably consistent, with each strain producing about 85% of the branched-chain fatty acids in the anteiso-form.
• strain G2 Comparing strain 3 to strain 5 (DV2 thrA*BC tdcB cimA3.7 leuBCD , also denoted "strain G2"), which is identical to strain 3 except for also overexpressing threonine deaminase, about 90% of the BCFA produced by strain 5 was in the anteiso-form, compared to about 84% in strain 3, which utilized the host cell's native threonine deaminase activity.
• strain 3 (Gl) and strain 5 (G2) indicates that engineering a microbial cell which is capable of producing branched chain fatty acids (owing to the presence of BKD and branched chain beta-ketoacyl-ACP synthase activities) to express or overexpress polypeptides having (R)-citramalate synthase activity,
• isopropylmalate isomerase activity and beta-isopropyl malate dehydrogenase activity (according to the (A.2) part of the anteiso-BCFA pathway) together with polypeptides having aspartokinase activity, homoserine dehydrogenase activity, homoserine kinase activity, threonine synthase activity, and optionally threonine deaminase activity (according to the (A.l) part of the anteiso-BCFA pathway) not only results in the production of compositions comprising anteiso-branched chain fatty acids, but compositions in which over 80% of the branched fatty acids produced are in the anteiso-form.
• E. coli strain DV2 (Example 1) is transformed with plasmids pKZ4 (expressing P. putida bkd genes), pDG6 (expressing B. subtilis fabHl), and a plasmid which expresses the ester synthase polypeptide Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021; SEQ ID NO:308)
• a polynucleotide encoding ES9 is synthesized by DNA2.0 (Menlo Park, CA), is subjected to restriction digestion with BspHI and Xhol, and cloned into plasmid pOP80 (Example 4) also digested with BspHI and Xhol, resulting in a plasmid expressing ES9 under the control of the P Trc promoter.
• About 31% of the FAM E produced were branched FAM E.
• About 74% of the branched FAME were iso-branched FAM E and about 26% were anteiso-branched FAME (Table 14).
• the branched chain FAEE detected included iso-C 2 Ci 2:0 , iso-C 2 Ci 3:0 , anteiso-C 2 Ci 3: o, iso-C 2 Ci 4: o, iso-C 2 Ci 5:0 , anteiso- 2Ci5:o, iso-C 2 Ci6:o, iso-C 2 Ci 7:0 and anteiso-C 2 Ci 7:0 ethyl esters.
• About 22% of the FAEE produced were branched FAEE.
• About 81% of those branched FAEE were iso-branched FAEE, and about 26% were anteiso-branched FAEE (Table 14).
• hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021) produce branched fatty esters.
• a polynucleotide sequence encoding the ES9 ester synthase polypeptide (SEQ ID NO:308) is cloned into B. subtilis expression vector pHTOl (MoBiTec GmbH, Goettingen, Germany ).
• Vector pHTOl is an Escherichia coli - Bacillus subtilis shuttle vector that carries the strong promoter Pgrac for protein expression in B. subtilis.
• the ES9 coding sequence is inserted between the BamHI and Xbal cloning sites.
• subtilis strain 1HA01 [lacA::spec leuB8 metB5 r(-)m(+) Sp ; obtained from Bacillus Genetic Stock Center, Columbus, OH, Strain Number BGSC 1A785) is transformed with pHT01_ES9 according to the protocol of
• B. subtilis 1HA01 cells are grown at 37°C in the miminal medium as described in Anagnostopoloulos and Spizizen (supra), supplemented with 50 ⁇ g/mL methionine
• B. subtilis transformed with the pHT01_ES9 vector produces branched methyl esters (when the culture is supplemented with methanol) or branched ethyl esters (when the culture is supplemented with ethanol), including branched esters of C13, C15 and C17 chain lengths.
• Example 9 Production of Branched Alkanes in E. coli
• Branched alkanes are produced by a recombinant microbial cell of the invention which expresses polynucleotides encoding polypeptides having fatty acid derivative enzyme activity, wherein the fatty acid derivative enzyme activity is hydrocarbon biosynthesis activity.
• the following example demonstrates the production of branched alkanes by a strain which expresses a polypeptide having acyl-ACP reductase (AAR) activity and a polypeptide having aldehyde decarbonylase (ADC) activity.
• AAR activity converts the branched acyl-ACP intermediate to a branched aldehyde
• ADC activity converts the branched aldehyde to a branched alkane.
• the Synechococcus elongatus PCC7942 aar gene, which encodes a fatty acyl-ACP reductase (GenBank Accession No. YP_400611; SEQ ID NO: 309) was integrated into E.coli strain MG1655 ⁇ /ac/E ⁇ (strain DV2; Example 1) to produce strain MG1655 ⁇ /adE htonA AAR:kan as follows: A polynucleotide encoding the aar gene controlled by a P trc promoter and flanked by a partial lacl gene and a kanamycin resistance cassette was amplified from plasmid pSL67-78A (SEQ ID NO:315) using primers AAR_F (5'-GGCT GGCTGG CATAAAT ATCTC-3'; SEQ ID NO:310) and AAR_R (5'- GTTATGATAT GTTGGTCGGATA AGCGTCGCGCCGCA
• SL106A The resulting strain was named SL106A.
• Strain SL106A was transformed with plasmid pTB38, which encodes aldehyde decarbonylase (ADC) from Nostoc punctiforme PCC73102 (GenBank Accession No. YP_001865325; SEQ ID NO: 312) under the control of the P trc promoter and contains a spectinomycin resistance cassette.
• ADC aldehyde decarbonylase
• the strain was then transformed with plasmids pKZ4 (expressing P. putida bkd genes) and pKZ5 (expressing B. subtilis fabHl) and evaluated for branched alkane production.
• the shake flask protocol using Che-9 media was followed.
• 1 mL of culture was extracted with 0.5 mL ethyl acetate (containing 1% HCI) and subjected to GC/MS analysis.
• a variety of straight chain and branched alkanes were produced (Table 15).
• the branched alkanes detected included iso-C14:0, anteiso-C14:0, iso-C16:0, and anteiso-C16:0 alkanes.
• About 14% of the alkanes produced were branched alkanes.
• About 54% of the ranched alkanes were iso-branched alkanes, and about 46% were anteiso-branched Ikanes (Table 15).
• the Synechococcus elongatus PCC7942 aar gene which encodes a fatty acyl-ACP reductase (GenBank Accession No. YP_400611; SEQ ID NO: 309) was integrated into the chromosome of E.coli strain MG1655 AfadE_AtonA as described in Example 9.
• the resulting E.coli strain MG1655 AfodE_AtonA AAR:kan was transformed with with plasmids pKZ4 (expressing P. putida bkd genes) and pDG6 (expressing B. subtilisfobHl). The strain was evaluated for production of branched chain alcohols using shake flask fermentation.
• Figure 7(A) shows that iso-branched and anteiso-branched C14-C17 fatty alcohols were produced by the recombinant microbial strain expressing a fatty acyl-ACP reductase (AAR), a branched chain alpha-keto acid dehydrogenase (BKD) complex, and a branched chain- specific ⁇ -ketoacyl-ACP synthase III (FabH).
• Figure 7(B) shows that branched fatty alcohols were not produced by the recombinant microbial strain expressing AAR but not BDK nor the branched chain-specific FabH.
• Example 11 Identification and Quantification of Branched Fatty Acid Derivatives
• the instrument is an Agilent 5975B MSD system equipped with a 30 mx0.25 mm (0.10 ⁇ film) DB-5 column.
• the mass spectrometer is equipped with an electron impact ionization source. Two GC/MS programs were utilized.
• GC/MS program #1 The temperature of the column is held isothermal at 90 g C for 5 min, then is raised to 300 9 C with a 25 5 C/min ramp, and finally stays at 300 ⁇ C for 1.6 min. The total run time is 15 min. With this program, the inlet temperature is hold at 300 ⁇ C.
• the injector is set at splitless mode. 1 ⁇ of sample is injected for every injection.
• the carrier gas (helium) is released at 1.0 mL/min.
• the source temperature of the mass spectrometer is held at 230°C.
• GC/MS program #2 The temperature of the column is held isothermal at 100 9 C for 3 min, then is raised to 320 g C with 20 9 C/min, and finally stays isothermal at 3202C for 5 min. The total run time is 19 min.
• the injector is set at splitless mode. 1 ⁇ of sample is injected for every injection.
• the carrier gas (helium) is released at 1.2 mL/min.
• the ionization source temperature is set at 230 g C.
• Extracts containing branched fatty acids, branched fatty acid derivatives, and/or branched alkanes produced by the engineered E.coli strains were analyzed on GC/MS. As described in Example 7 above, various branched-chain fatty acids, fatty acid derivatives, such as fatty esters, and branched alkanes were detected.
• a mixture of bacterial acid methyl ester (BAME, Sigma-Aldrich, Cat #: 47080-U 10 mg/mL total concentration) contains the following 26 compounds:
• the data sheet which provides the GC eluting sequence of all 26 components in the BAME mixture was obtain from a SPB-1 phase column.
• the retention time (RT) of these compounds analyzed with these two GC programs are listed in the table below. These retention times were used to identify the branched compounds produced by the recombinant microbial strains.
• branched fatty acid branched fatty acid derivatives e.g., branched fatty acids, alcohols, esters and hydrocarbons
• Standard curve quantitation method was applied, wherein standard mixtures with different concentrations were analyzed by the same GC/MS program as the samples. After data acquisition, the instrument response (total ion current) was plotted against the concentrations of the standards. Linear calibration curves were obtained.
• Table 18 lists the fatty methyl esters used as standards to quantify various branched fatty acid methyl esters.
• Table 19 lists fatty ethyl esters used as standards to quantify various branched fatty acid ethyl esters.
• CiCi 4:0 fatty acid methyl ester was used as a standard for quantitating derivitized branched free fatty acids in the extract from any production strain (Table 21). The measured concentrations were then converted back to branched free fatty acid concentrations based on their molecular weights.
• Branched alkanes were measured using the following standards (Table 22), which were also used to verify the amount of branched fatty aldehydes or branched fatty alcohols. Table 22

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